Azulenyl nitrone spin trapping agents, methods of making and using same

ABSTRACT

The present invention relates to chromotropic nitrone spin trapping agents, methods of making these agents, compositions comprising same, and methods of their use. In particular, azulenyl nitrones of the present invention are effective agents for trapping free radical species and find use as efficient antioxidants in physicochemical and biological systems. Accordingly, the invention also relates to spin adducts formed from the combination of azulenyl nitrones with free radicals. The compounds of the present invention are readily prepared from available starting materials and find further use in assays and in a number of diagnostic, prophylactic and therapeutic applications, including but not limited to the alleviation, modulation and inhibition of the negative effects of carbon-centered or oxygen-centered radical species and other products of oxidation. Moreover, the combination adducts may be calorimetrically detected and, optionally, isolated and characterized to obtain valuable information (e.g., structural and the like) about the original reactive free radical species.

REFERENCE TO RELATED APPLICATION

This Application is a continuation of International Application No.PCT/US96/18570, whose international filing date is Nov. 15, 1996, whichin turn claims the benefit of U.S. Provisional Patent Application No(s).60/006,949 and 60/024,631, filed Nov. 17, 1995 and Aug. 27, 1996respectively, the disclosures of which Applications are incorporated byreference herein. The benefit of the filing and priority dates of theInternational and United States Applications is respectfully requested.

1. FIELD OF THE INVENTION

The present invention relates to chromotropic azulenyl nitrone spintrapping agents, methods of making these agents, compositions comprisingsame, and methods of their use. In particular, azulenyl nitrones of thepresent invention are effective agents for trapping and identifying freeradical species and find use as efficient antioxidants inphysicochemical and biological systems.

2. BACKGROUND OF THE INVENTION

2.1. General Considerations

The technique of spin trapping is an important method for garneringinformation on free radicals difficult or impossible to detect by directspectroscopic observation due to their exceedingly short lifetimes andlow concentrations. To date, two classes of spin trapping agents havereceived the most attention, namely nitroso compounds and nitrones. Ofthese, the latter have been more frequently used, especially inbiological systems.

The most commonly cited drawbacks to the application of spin trappingagents bearing nitroso functionality are instability and toxicity. Onaccount of these undesirable characteristics, researchers often opt fornitrone spin traps despite the fact that their nitroxide spin adductsgenerally provide less structural information from ESR than do thosefrom nitroso-based spin traps. Furthermore, the nitroxides obtained fromthe addition of certain carbon-centered radicals (tertiary alkyl andaryl) to the most widely used nitrone spin trapsalpha-phenyl-N-tert-butylnitrone (PBN), pyridineN-oxide-4-N-tert-butylnitrone (POBN) and dimethylpyrroline N-oxide(DMPO) are, due primarily to disproportionation, less persistent thanthose obtained from addition of such radicals to nitroso compounds.

Several groups have described the use of isotopically labeled spin trapsor the application of special equipment consisting of GC/MS orHPLC-interfaced ESR spectrometers designed to detect, isolate andcharacterize free radical adducts of nitrone spin traps in biologicalsystems with varied success.

2.2. Detection and Characterization of Free Radicals

Nitrones behave as spin trapping agents when a diamagnetic nitronecompound (the "spin trap") reacts with a transient free radical species(having the "spin") to provide a relatively more stable radical species(referred to as the "spin adduct"). The spin adduct may be detectable byelectron paramagnetic resonance (EPR) spectroscopy if the spin adducthas a reasonable lifetime. Thus, information about the spin can begleaned from a study of the structure and spectroscopic characteristicsof the spin adduct. For example, the toxicity of synthetic beta-amyloidpeptide preparations toward glutamine synthetase could be correlatedwith the characteristics of the EPR signal generated by the spin adductformed from each batch of synthetic beta-amyloid peptide and the spintrap PBN. See, Hensley, K. et al., in NeuroReport (1995) 6:489-492.Beta-amyloid peptides are neurotoxic substances that are postulated tobe involved in the etiology of Alzheimer's disease.

2.3. Methods of Diagnoses

Low molecular weight nitroxides are non-immunogenic. Moreover, they aretypically cell permeable and can exist as a non-toxic, stable freeradical capable of partitioning among various cellular compartments.Being paramagnetic, nitroxides are detectable by electron paramagneticresonance (EPR) spectrometry and may serve as contrast agents inmagnetic resonance imaging (MRI). See, Brasch, R. C., in Radiology(1983) 147:781; Keana, J. F. and Van, N. F., in Physiol. Chem. Phys.Med. NMR (1984) 16:477. Nitroxides have also been used as biophysicalmarkers to probe cellular metabolism, oxygen level, intracellular pH,protein/lipid mobility and membrane structure. Hence, nitroxides finduse in a number of diagnostic methods to determine thephysiological/medical condition of a subject or the biophysicalcharacteristics of a given sample, including samples obtained from abiological fluid.

2.4. Therapeutic Applications of Spin Trapping Agents

Free radicals and oxidative damage have been implicated in brain agingand several neurodegenerative diseases. See, Socci, D. J. et al., inBrain Research (1995) 693(1-2):88-91. Chronic treatment of aged ratswith certain compounds, including the spin trapping agent alpha-phenylN-tert-butylnitrone (PBN) and the antioxidant alpha-tocopherol (vitaminE), was found to benefit (i.e., improve) age-related changes incognitive performance.

In vitro and in vivo evidence is mounting that the administration ofantioxidants can strongly reduce the rate of progression of lesionformation associated with the process of atherosclerosis. Based onseveral experimental models, including low density lipoprotein(LDL)-receptor-deficient rabbits, cholesterol-fed rabbits andcholesterol-fed non-human primates, several antioxidants have manifesteda 50-80% reduction in the rate of progression of lesions. Theeffectiveness of probucol, butylated hydroxytoluene (BHT),N,N'-diphenylphenylenediamine and vitamin E are attributed to theirrespective antioxidant potentials and to the proposition that oxidativemodification of LDL contributes to the progression of atherosclerosis.See, Steinberg, D., in Lancet (1995) 346(8966):36-38. The one-electronoxidative potentials (vs. NHE) of vitamin E in an aqueous solution at pH7 and 20 degrees C. is 0.48 V. The oxidative potentials of PBN, POBN andDMPO range from about 1.5-2.0 V.

Further, Downs, T. R. et al., in Int'l J. Immunopharmacol. (1995)17(7):571-580, have shown that a cyclic nitrone antioxidant, MDL101,002, reduces organ dysfunction and cytokine secretion induced bylipopolysaccharide (LPS) administered to rats. These authors also testedthe ability of MDL 101,002 to prevent LPS-induced pulmonary edema,leukopenia and thrombocytopenia. They found that MDL 101,002 preventedpulmonary edema, partially reduced thrombocytopenia but failed toprevent leukopenia. These workers found that their results wereconsistent with the role that oxygen free radicals played in thedevelopment of endotoxin-induced organ dysfunction and shock. Theyfurther suggest that free radical scavengers could reduce the mortalityconsequent to sepsis by organ dysfunction, at least in part, through areduction in free radical-stimulated cytokine secretion.

2.4.1. Radicals in Allergy and Allograft Rejection

Allergic reactions generate reactive oxygen species, includingsuperoxide anions, which usher the influx of inflammatory cells to thesite of allergen challenge and contribute to allergic inflammation. Theinflammation may, in turn, lead to cell or tissue injury. For allergicreactions in the lung, these processes are also accompanied by increasedvascular permeability and changes in airway mechanics. See, Sanders, S.P. et al. in Am. J. Respir. Crit. Care Med. (1995) 151:1725-1733. Thus,the administration of spin trapping agents to the site of challenge mayreduce the inflammatory response and help reduce tissue or cell damage.

Separately, oxygen-derived free radicals are suspected in playing a rolein cytotoxicity during episodes of allograft rejection/destructionfollowing infiltration of the graft by mononuclear cells. Theadministration of radical scavengers may thus inhibit or reduce theincidence of allograft rejection. See, Roza, A. M. et al., inTransplantation Proceedings (1994) 26(2):544-545.

New reagents that could visually signal the formation of oxidativespecies would be extremely useful not only in skin tests or in cellculture but also in determining, for example, the compatibility of apatient's white blood cells with a particular kidney dialysis membrane.In vitro calorimetric assays would be of great utility.

2.5. Other Applications

PBN has been shown to offer protection in the cardiovascular diseasearea, in particular, by trapping free radicals generated duringischemia-reperfision-mediated injury to the heart. See, e.g., Bolli, R.et al. J. Clin. Invest. (1988) 82:476. The benefits of trapping freeradicals generated in similar types of injury to the brain ofexperimental animals has also been demonstrated. See, e.g., Oliver, C.N. et al. Proc. Nat'l. Acad. Sci. USA (1990) 87:5144; Carney, J. M. etal. Ibid. (1991) 88:3636; Floyd, R. A. Science (1991) 254:1597.Oxidative damage to protein and DNA is mediated by oxygen free radicalintermediates, leading to strand breaks and base modifications. Enzymes,such as glutamine synthetase, can also be inactivated by oxidativeprocesses. Such damage can be observed, for example, in animalssubjected to brain ischemnia/reperfusion injury. See, Floyd, R. A. andCarney, J. M. Ann. Neurol. (1992) 32:S22-S27.

Evidence is also available that PBN inhibits oxidative modification ofcholesterol and triglycerides of Low Density Lipoproteins (LDL).Oxidative modification of LDL, along with lipid peroxidation andfree-radical mediated reactions, is a process that is implicated in theinitiation of atherosclerosis. See, e.g., Steinberg, D. et al. N. Engl.J. Med. (1989) 320:915; Esterbauer, H. et al. Ann. N. Y. Acad. Sci.(1989) 570:254.

2.5.1. Life Span Extension and Delay of Onset of Senescence

Free radicals and oxidative damage have been proposed as the underlyingreasons for aging, chronic and degenerative diseases of aging, and acuteclinical conditions. Daily administration by intraperitoneal injectionof PBN to an aged animal model showed that PBN offered a remarkableextension of the lifespan in both male and female populations. See,Packer, L. et al., in Biochem. Biophys. Res. Commun. (1995)211(3):847-849. These authors conclude that PBN could have prophylaticvalue against the onset of, at least, pathological senescence.

Bruce N. Ames and co-workers, in an article published in the Proc.Nat'l. Acad. Sci. USA (1995) 92:4337-4341, found support for thehypothesis that oxidative DNA damage contributes to replicativecessation in human diploid fibroblast cells. These workers found thatsenescent cells, those cells that have ceased growth in culture after afinite number of population doublings, excise from DNA four times more8-oxoguanine per day than do early-passage young cells. Also, levels of8-oxo-2'-deoxyguanosine in DNA of senescent cells are about a thirdhigher than those found in DNA of young cells. Most interestingly, theyfound that PBN, perhaps acting as either an antioxidant or as a spintrapping agent, effectively delayed the onset of senescence andrejuvenated near senescent cells.

2.6. Applications as Food and Fuel Additives

2.6.1. Quality Evaluation of Fats

A number of factors influence fat stability and the formation of lipidoxidation products. Increased unsaturation, increased frying time,increased exposure of the oil to air and increased trace metal contentwill all result in decreased oxidative stability. The presence ofsilicones in a frying oil will cause increased oil stability by yetunknown mechanisms. Published data indicates that filtration of oilsthrough certain active adsorbents will increase the useful frying lifeof an oil during actual fryer use by removal of colored materials, freefatty acids and other oxidation products.

Usually peroxides decompose at about 150° C. Therefore at fryingtemperatures, the accumulation of peroxides does not occur. Peroxidevalues usually are a measure of lipid oxidation at lower temperaturessuch as those used for storage of fats or a product. The relationshipbetween storage time and peroxide value can then be used to measurequality.

The Schall oven test involves simply putting a small amount of the fatinto a beaker and placing it into an oven under standardized conditionsat 60° C. to oxidize the sample. Samples are then taken and peroxidevalues determined on them. There are many other tests available to checkfrying oil quality, all which purport to tell the operator when to dosomething with the used fat--either filter it through active filters,discard it, or dilute it with a less degraded fat. Some tests which havebeen used to check frying oil quality are the saponification colorindex, 2,6-dichloroindole phenol color test, methylene blue color test,and iodine color scale. These tests allegedly determine when the fat hasgone bad and can no longer produce a high quality food product. Forinstance, the Rau test from E. Merck is a colormetric test kit whichcontains redox indicators that react with total oxidized compounds in asample. It has a four color scale and is used for diagnoses of fatquality. The fourth color scale indicates a bad oil and the oil shouldbe discarded. All these tests differ in reliability and may be moretedious to perform than necessary.

2.6.2. Gasoline Storage and Antioxidants

Surprisingly, difficulty in starting a lawn mower, trail bike, outboardmotor, or similar infrequently used gasoline engine, is caused by "bad"petroleum. Petroleum is subject to autoxidation, like oils in foods andin the human body. When gasoline is left for any long period (e.g., afew months or more), gums are formed by the reaction of oxygen withunsaturated components of the fuel. BHT (also known as 2,6-di-tert-butylp-cresol) is a U. S. Government approved gasoline additive that meetsmilitary requirements for gasoline stability. A half pound of BHT addedto 1,100 gallons of gasoline prevents gum formation when gasoline wasstored in sealed (with standard rubber washers) 5-gallon cans forperiods up to two years in the Mojave desert in full sunlight, comparedto a storage life of only a few months for unprotected gasoline. Theamount currently recommended for military use is 1 pound BHT to 1,100gallons of gasoline. For even longer storage, BHT, alone, may not beenough to prevent spoiling of the fuel.

Other materials besides fuels that are affected by similar agingmechanisms include plastics, rubber, paints, asphalt, roofing shingles,oils and lubricants.

Accordingly, there exists a continuing need to discover new, effectivesubstances exhibiting free radical/spin trapping and/or antioxidantactivity which are potentially useful for a wide range of analytical,preservative, diagnostic, prophylactic and therapeutic applications.

3. SUMMARY OF THE INVENTION

Accordingly, the present invention provides a new class of nitrone spintrapping agents, namely azulenyl nitrones, which can be efficientlyprepared from abundant sesquiterpenes or their synthetic analog. Theazulenyl nitrones of the present invention possess the unprecedentedcapacity to tag free radicals by yielding characteristically colored andhighly visible diamagnetic (and paramagnetic) spin adducts. For example,spin trapping of 1-cyanocyclohexyl radicals (generated by thermaldecomposition of the corresponding azo compound in toluene) with a greenazulenyl nitrone 1 (Nu=OEt, below) produces a violet double spin adduct(via 1,3-addition of two 1-cyanocyclohexyl radicals to the nitronemoiety). The double spin adduct, by virtue of its characteristic,visible chromophore, is easily detected and purified.

The obvious green to violet chromotropism that accompanies conversion ofthe nitrones of the invention to commonly formed diamagneticdecomposition products of intermediate nitroxide spin adducts (which arestill covalently attached to the radical units of interest) render suchnitrones useful in tracking free radical residues, especially infrequently encountered cases involving fast annihilation of paramagneticnitroxide spin adducts via either combination, disproportionation orreduction.

Vivid chromotropism (green to red) has also been observed in aerobiclipid peroxidation studies with azulenyl nitrones in corn oil and pointsto potential applications of these novel nitrones as indicators ofoxidative stress in lipids or as preservatives in such lipids or othercompositions susceptible to oxidative breakdown, including otherfoodstuffs and fuels. The azulenyl nitrones of the invention are alsouseful for alleviating a host of ill effects caused generally byreactive free radicals or oxidative processes in biological systems.

Therefore, it is an objective of the present invention to provide acompound of the general formula, below, ##STR1##

in which R₁ may be a hydrogen, a linear or branched alkyl groupcomprising 1-6 carbon atoms, or an aryl group comprising 6-10 carbonatoms; R₂ may be a linear or branched alkyl group comprising 1-6 carbonatoms, or an aryl group comprising 6-10 carbon atoms; R₃ may be ahydrogen, or a linear or branched allyl group comprising 1-6 carbonatoms; R₄ may be a hydrogen, or a linear or branched allyl groupcomprising 1-6 carbon atoms; R' may be a linear or branched alkyl groupcomprising 1-6 carbon atoms; W may be a linear or branched alkyl groupcomprising 1-6 carbon atoms, an aryl group comprising 6-10 carbon atoms,or an electron-withdrawing group; n may be 0, 1, or 2 (if n is 2, each Wmay be the same as or different from one another); m may be 0, 1, 2, or3 (if m is 2 or 3, each R' may be the same as or different from oneanother); o may be 1 or 2 (if o is 2, each R₁ and R₂ may be the same asor different from one another); p may be 0, 1, or 2 (if p is 2, each R₃and R₄ may be the same as or different from one another) or a saltthereof.

In specific embodiments of the invention, a compound is contemplated inwhich o is 1, p is 0, n is 1, m 1 or 2, or in which the groups R₁, R₃,and R₄ are all hydrogen, in which at least one R' is a methyl, ethyl, orisopropyl group, or in which the group R₂ is a tert-butyl group. Ofparticular interest are compounds in which W is an electron-withdrawinggroup.

In other embodiments, the compound of interest bears the group (CR₃═CR₄)_(p) C(R₁)═N⁺ (R₂)O⁻ at the 1-position of the azulene ring systemwhen the group W is at the 3-position. In still others, m is 2 and thegroups R' are at the 4- and 7-positions. Further, the group W may be acarboxylic acid, carboxylic acid ester, sulfonic acid, sulfonic acidester, ketone, halogen, cyano, nitro, nitroso, aldehyde, phosphoricacid, phosphoric acid ester, sulfoxide, sulfone, or a salt thereof. Inpreferred cases, W is a carboxylic acid, sulfonic acid, or their salts,or a trifluoroacetyl group.

In terms of stereochemistry, the R₃ and R₄ groups of the generalformula, as well as the R₁ and R₂ groups of the general formula, may becis or trans to one another. Preferably, the R₃ and R₄ groups are transto each other and the R₁ and R₂ groups are cis to one another. The R₅and R₆ groups of the general formula, may be cis or trans to oneanother, preferably, trans to each other.

Certain compounds are preferred, including 2-methyl[1-(3-carboxylicacid-7-isopropyl-4-methyl)azulenylmethylene]-2-propanamine N-oxide;2-methyl[1-(3-carboethoxy-7-isopropyl-4-methyl)azulenylmethylene]-2-propanamineN-oxide; 2-methyl[1-(3-sulfonicacid-7-isopropyl-4-methyl)azulenyhmethylene]-2-propanamine N-oxide;2-methyl[1-(3-methylsulfonyl-7-isopropyl-4-methyl)azulenylmethylene]-2-propanamineN-oxide; 1,3-bis(2-methyl-2-propanamine N-oxide)azulenyldimethylene; and1,3-bis(2-methyl-2-propanamineN-oxide)-7-isopropyl-4-methylazalenyl-dimethylene. Each compound of theinvention includes its acid, ester, amide, salt, or crystalline form, asappropriate.

The invention also contemplates a variety of methods, including but notlimited to, a method of trapping a reactive free radical comprisingproviding a compound of the invention and allowing the compound tocombine with a reactive free radical to provide an adduct comprising thefree radical and the compound, a method of detecting oxidation productsin a medium comprising combining or contacting a compound of theinvention with a medium and detecting the presence of an adduct or anend-product thereof in the resulting mitre, a method of alleviating theill effects of a pathologic condition mediated or initiated by areactive free radical comprising administering an effective amount ofthe compound of the invention to a subject in need thereof. Still othermethods include, but are not limited to, methods of alleviating,ameliorating, treating, preventing, managing, or inhibiting the negativeeffects of ischemia, reperfusion injury, trauma (particularly head orbrain trauma), acute respiratory distress syndrome, neurological(especially cerebral) disorders, Alzheimer's disease, stroke,Parkinson's disease, Huntington's disease, Lou Gehrig's disease,Wilson's disease, aging, senescence, apoptosis, inflammation and thelike.

The invention further contemplates compositions comprising a compound ofthe invention and an appropriate carrier, especially compositions havingpharmaceutical, dermatological, cosmetic, or industrial applications.

It should also be apparent that the invention is further directed to aspin adduct comprising a combination product of an azulenyl nitrone anda free radical.

Likewise, it is also an object of the invention to provide a process formaking an azulenyl nitrone comprising: (a) providing an azllene; (b)introducing a acyl group to the azulene at a position that is to bear anitrone group; and (c) converting the acyl group to a nitrone group toprovide an azulenyl nitrone.

Other objects of the present invention, including compositions andmethods of using the azulenyl nitrones of the invention will be apparentto one of ordinary skill considering the detailed descriptions providedherein.

4. BRIEF DESCRIPTION OF THE FIGURE

FIGS. 1A and 1B illustrates the three-line ESR signal generated by thenitroxide spin adduct formed by the combination of azulenyl nitrone, 1(Nu=OEt) and the free radical derived from the thermolysis of AMVNazulenyl nitrone, Compound 1.

5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Herein is reported a new and simple colorimetric approach to thedetection, isolation and analysis of free radical adducts of nitronesemploying the novel nitrone spin trapping agents, such as 1 (Nu=OEt),which are easily obtained from azulene and its derivatives, includingthe abundant sesquiterpene, guaiazulene. ##STR2##

Of particular importance regarding spin trapping with the azulenylnitrones of the present invention is their capacity to tag free radicalsby yielding characteristically colored and highly visible diamagnetic(and paramagnetic) spin adducts. Thus, nitrones, such as 1 (Nu=OEt), orany of the other nitrones contemplated herein, provide the potential toimplicate the intermediacy and establish the identity of free radicalsin situations in which presently available ESR detection/isolationtechnology may fail.

Albeit considerably more persistent than most free radicals, nitroxidesare nevertheless often subject to the usual free radical destructionprocesses of combination, disproportionation, and oxidation/reduction,yielding diamagnetic products. The rapid formation of diamagnetic spinadducts in traditional spin trapping experiments is an unwantedoccurrence which can constitute a serious obstacle, because once suchproducts are formed in biological systems employing conventional nitronespin traps, they are lost amidst a vast number of diverse diamagneticmolecules.

The ability to easily locate diamagnetic spin adducts in complexmixtures offers an appealing alternative should one be faced withtechnical difficulties often encountered while attempting to isolatenitroxides resulting from conventional nitrone spin traps before theydecay into diamagnetic species. In spin trapping with the compounds ofthe present invention, the characteristic chromophore of the diamagneticspin adducts arising from nitroxides via combination, disproportionationor reduction, while crucially different from the chromophore of theazulenyl nitrone, is in fact the same as that of the initially formedESR-detectable nitroxide spin adducts. Therefore, this characteristicchromophore should also expedite the purification (and subsequentstructure determination) of these paramagnetic species from reactionmixtures amenable to nitroxide longevity.

Even though nitroxides possess a visible chromophore of their own, theircharacteristic red color is due to an absorption with a very lowextinction coefficient centered around 460 nm. For example, the visibleabsorption spectrum in hexane for di-t-butylnitroxide shows a maximum at465 nm with log e=0.95. The extinction coefficient for the absorptiongiving rise to the color of the diamagnetic azulene-containing spinadducts described herein is between one to two orders of magnitudegreater. See, Smith, P. A. S. Open-Chain Nitrogen Compozinds, W. A.Benjamin, Inc., New York, 1965, Vol. 2, p. 105 and references citedtherein for additional discussions on nitroxide absorption spectra.

5.1. Preparation of Azulenyl Nitrones

Azulenyl nitrones of the present invention are prepared readily from avariety of available starting materials. For example, the azulenylnitrone, 1 (Nu=OEt) a stable green solid (mp 43-45° C.), is readilyprepared in three steps from guaiazulene (Scheme I, below). Exposure ofguaiazulene to oxalyl bromide in ether at room temperature according tothe method of Treibs, W. et al., described in Chem. Ber. (1959) 92:2152,yields acyl bromide 2, which is directly esterified with EtOH to providethe violet ethyl ester 3 in 80% yield. Oxidation of 3 with twoequivalents of DDQ in aqueous acetonitile at room temperature in analogyto the method of Takase (See, Amemiya, T. et al., in Chem. Lett. (1977)587) affords a 74% yield of red aldehyde 4.

Condensation of 4 with N-tertbutylhydroxylamine hydrochloride inpyridine at 95° C. provides 1 (Nu=OEt) in nearly quantitative yield.Spectral data for 1 (Nu=OEt): ¹ H NMR (300 MHz, CDCl₃): 9.74(s, 1H),8.36(s, 1H), 8.17(s, 1H), 7.54(d, J=11 Hz, 1H), 7.43(d, J=11 Hz, 1H),4.37(q, J=7 Hz, 2H), 3.17(m, 1H), 2.97(s, 3H), 1.71(s, 9H), and1.38-1.43(m, 9H). ¹³ C NMR (75.4 MHz, CDCl₃): 167.2, 148.7, 145.8,141.3, 141.0, 137.7, 136.8, 132.7, 132.5, 123.0, 120.7, 117.1, 69.6,60.8, 38.3, 28.4, 27.9, 24.4, and 14.4. IR (neat): 3135, 2965, 2930,2905, 2870, 1715, 1580, 1560, 1460, 1335, 1300, 1245, 1195, 1150, 1105,1040, 920 cm⁻¹. UV-VIS max (hexane): 313 nm (ε=26,071), 358 (15,526),417 (8,390), and 588 (532). Exact Mass (FABMS, NBA); Calculated for C₂₂H₃₀ NO₃ (M⁺ +1): 356.2226. Found: 356.2230. ##STR3##

5.1.1. Preparation of Bis-Nitronyl Azulene

Bis-nitrones are also readily prepared by the methods of the invention.In particular, the bis-nitronyl azulene 34, a water soluble dark greencrystalline material (mp 211-212° C.), is readily prepared from thebis-aldehyde, 1,3-azulenedicarboxaldehyde. The bis-aldehyde is preparedaccording to the method of Hafaer, K. and Bernhard, C., Annalen (1959)625:108. The bis-nitrone is prepared as follows. ##STR4##

1,3-Azulenedicarboxaldehyde (600 mg) is dissolved in 6.5 ml of pyridine.Magnesium sulfate (1200 mg) and N-tert-butylhydroxylammne hydrochloride(1638 mg,) is added to the solution,. The mixture is heated wit stirringto 95° C. under nitrogen and is stirred for 13 hours. Upon cooling tort, the reaction mixture is poured into a separator flinnel containing60 ml of CHCl₃ and 60 ml of sat aq. NaHCO₃. The aqueous layer isseparated and washed with three 30 ml portions of CHCl₃. The combinedorganc layers are dried over anhydrous MgSO₄, filtered, and evaporatedto give the bis-nitrone 34 (940 mg, 89% yield) as dark green crystals.Oxidation potential equals 0.72 V vs SCE. ¹ H NMR (CDCl₃): 10.35 (s,1H), 8.65 (d, 2H, J=10 Hz) 8.11 (s, 2H), 7.68 (t, 1H, J=10 Hz), 7.32 (t,2H, J=10 Hz), 1.67 (s, 18H). ¹³ C NMR (CDCl₃): 139.9, 135.0, 134.2,125.8, 125.6, 123.2, 120.5, 69.8, 28.4. IR (thin film): 3052, 2972,1644, 1564, 1452, 1404, 1356, 1261, 1196, 1124, 1052, and 892 cm⁻¹.

5.1.2. Preparation of Conjugated Azulenyl Nitrones

Coupling azulenyl nitrones such that they are in electronic conjugationreduces their oxidation potential and thus forms a more reactive spintrapping agent. Electronically conjugated azulenyl nitrones are preparedfrom a conjugated azulenyl chain. Conjugated azulene chains are made bycoupling azulene carboxaldehydes that, in turn, are prepared by avariety of methods. Bis-aldehydes are prepared as described in Section5.1.1. Azulenes with a single aldehyde substituent is preparedsimilarly.

For example, guiazulene is converted to its aldehyde by treatment withphosphorous oxychloride (POCl₃) in an excess of dimethylformamide toprovide guiazulene carboxaldehyde. The guiazulene carboxaldehyde isdimerized in high yield to an alkene-linked conjugated, unsaturatedsystem by treatment with titanium trichloride. The coupled product istreated with DDQ, then with magnesium sulfate andN-tert-butylhydroxylamine hydrochloride, as described in Section 5.1, togenerate the 1,2-bis(azulenyl nitrone)ethylene. A general formula is asfollows: ##STR5## in which s can be 0 or greater, preferably 1, 2, 3, 4,5, or up to 100. It should be apparent to those of ordinary skill, thata dimer can be prepared or a polymer having three, four, five, or manymore azulenyl units, by the methods disclosed in the present invention.

Using the methods herein, any number of azulenyl nitrones can beprepared. Accordingly, the following representative compounds, includingtheir salts (especially their alkali and alkaline-earth metal salts ortheir acetic and hydrochloride acid addition salts) are obtained by themethods of the present invention:

#1 2-methyl[1-(3-carboethoxy-7-isopropyl-4-methyl)azulenylmethylene]-2-propanamine N-oxide; having an IUPAC name of(Z)-3-[[1,1-dimethylethyl)oxidoimino]methyl]-8-methyl-5-(1-methylethyl)-1-azulenecarboxylicacid, ethyl ester (CAS Registry No. 174355-72-7)

#2 2-methyl[1-(3-dimethylamido-7-isopropyl-4-methyl)azulenylmethylene]-2-propanamine N-oxide

#3 2-methyl[1-(3-formyl-7-isopropyl-4-methyl)azulenylmethylene]-2-propanamine N-oxide

#4 2-methyl[1-(3-carboxylic acid-7-isopropyl-4-methyl)azulenylmethylene]-2-propanamine N-oxide, sodium salt

#5 2-methyl[1-(3-trifluoroacetyl-7-isopropenyl-4-methyl)azulenylmethylene]-2-propanamine N-oxide

#62-methyl[1-(3-acyl-7-isopropyl4-methyl)azulenyl-methylene]-2-propanamineN-oxide, spermine conjugate

#7 2-methyl[1-(3-diethylamido-7-isopropyl4-methyl)azulenylmethylene]-2-propanamine N-oxide

#82-methyl[1-(3-acyl-7-isopropyl-4-methyl)azulenyl-methylene]-2-propanamineN-oxide,N-methyl(D) glutamine conjugate

#9 2-methyl[1-(3-octadecylamido-7-isopropyl-4-methyl)azulenylmethylene]-2-propanamine N-oxide

#102-methyl[1-(3-acyl-7-isopropyl-4-methyl)azulenyl-methylene]-2-propanamineN-oxide, sphingosine conjugate

#112-methyl[1-(3-acyl-7-isopropyl-4-methyl)azulenyl-methylene]-2-propanamineN-oxide, polylysine conjugate

#122-methyl[1-(3-acyl-7-isopropyl-4-methyl)azulenyl-methylene]-2-propanamineN-oxide, (dG)₁₀ conjugate

#132-methyl[1-(3-acyl-7-isopropyl-4-methyl)azulenyl-methylene]-2-propanamineN-oxide, anti-bovine IgG(mouse) mAB conjugate

#142-methyl[1-(3-acyl-7-isopropyl-4-methyl)azulenyl-methylene]-2-propanamineN-oxide, N-(3-aminopropyl)-9 acridinamine conjugate

#152-methyl[1-(3-acyl-7-isopropyl-4-methyl)azulenyl-methylene]-2-propanamineN-oxide, histone type II-AS conjugate

#16 2-methyl[1-(3-N-t-butyinitronyl)-7-isopropyl-4-methyl)azulenylmethylene]-2-propanarnine N-oxide

#17 2-methyl[1-(7-isopropenyl-4-methyl)azulenylmethylene]-2-propanamineN-oxide

#18 2-methyl[1-azulenylhethylene]-2-propanamine N-oxide

#19 2-methyl[6-azulenylmethylene]-2-propanamine N-oxide

#20 2-methyl[4-azulenylmethylene]-2-propanarine N-oxide

#21 2-methyl[4-(1-methyl-7-isopropyl)azulenylmethylene]-2-propanamineN-oxide

#222-methyl[6-(1,4-dimethyl-7-isopropyl)azulenyl-methylene]-2-propanamineN-oxide

#23 2-methyl[1-(3-carboethoxy-7-isopropyl-4-methyl)azulenylmethylene]-2-propanamine N-oxide

#242-methyl[1-(3-cyano-7-isopropyl-4-methyl)azulenyi-methylene]-2-propanamineN-oxide

#25 2-methyl[1-(3-methylsulfonyl-7-isopropyl-4-methyl)azulenylmethylene]-2-propanarnine N-oxide

#26 2-methyl[1-(3-sulfonic acid-7-isopropyl-4-methyl)azulenylmethylene]-2-propanamine N-oxide, sodium salt

#272-methyl[1-(3-dimnethylphosphonato-7-isopropyl-4-methyl)azulenylmethylene]-2-propanamineN-oxide

#28 2-methyl[1-(3-phosphondioxy-7-isopropyl-4-methyl)azulenylmethylene]-2-propanamine N-oxide, disodium salt

#29 2-methyl[1-(3-nitro-7-isopropyl-4-methyl)azulenylmethylene]-2-propanamine N-oxide

#30 2-methyl[1-(3-carboethoxy-7-isopropyl4-methyl)azulenylpropenylene]-2-propanamine N-oxide

#31 2-methyl[1-(7-acetyl-4-methyl)azulenylmethylene]-2-propanamineN-oxide

#32[1-(3-carboethoxy-7-isopropyl-4-methyl)azulenyl-methylene]benzenamineN-oxide

#33 2-methyl[1-(7-isopropyl-4-methyl)azulenylmethylene]-2-propanamnineN-oxide

#34 1,3-bis(2-methyl-2-propanarnine N-oxide)azulenyldimethylene

#35 1,2-bis(azulenyl nitrone)ethyene

Doubtless, other azulenyl nitrones, especially various metal, ammonium,or acid addition salts, not specifically listed above, will be apparentto those of ordinary skill in view of the present disclosure. Such otherazulenyl nitrones are considered to fall within the scope of the presentinvention, however.

5.2. Characteristics of the Azulenyl Nitrones

5.2.1. Adduct Formed with Free Radical Species

The obvious chromotropism that accompanies conversion of nitrone spintraps, such as 1 (Nu=OEt), to diamagnetic free radical adducts arisingvia either combination, disproportionation, or reduction of intermediatenitroxides is unprecedented and may render them useful in tracking freeradical residues in frequently encountered cases involving fastannihilation of nitroxide spin adducts via any of the aforementionedprocesses.

Thus, when an emerald green solution of azulenyl nitrone 1 (Nu=OEt) intoluene (60 mM) is heated to 95° C. in the presence of azo compound 5under argon (See, Scheme I, above), TLC analysis of the progress of thereaction reveals the formation of a violet product of lower polaritythan 1 (Nu=OEt). When the reaction mixture is poured onto a flashchromatography column containing chloroform-saturated silica gel andeluted with chloroform, a violet band descends and is easily collected.Further purification by preparative TLC (chloroform) affords the violetdouble spin adduct 6. Spectral data for 6: ¹ H NMR (300 MHz, CDCl₃):8.74(s, 1H), 8.20(s, 1H), 7.57(d, J=11 Hz, 1H), 7.36(d, J=11 Hz, 1H),4.57(s, 1H), 4.39(q, J=7 Hz, 2H), 3.13, (m,1H), 3.02(s, 3H),1.53-2.78(m, 20H), 1.35-1.49(m, 9H), and 1.14(s, 9H). IR (neat): 2960,2940, 2860, 2220, 2200, 1705, 1415, 1260, 1195, 1095, 1040, and 800cm⁻¹. UV-VIS max (hexane): 301 nm (ε=10,209), 351 (2,097), 370 (2,558),and 548 (198). Exact Mass (FABMS, NBA); Calculated for C₃₆ H₅₀ N₃ O₃ (M⁺+1): 572.3852. Found: 572.3853.

5.2.2. Competitive Spin Trapping Behavior

An inspection of the ¹ H NMR spectrum of the reaction mixture formed ina competitive spin trapping experiment entailing therrnolysis (95° C., 6h) of a toluene solution containing 100 mM concentrations of 1 (Nu=OEt),PBN, and azo compound 5 indicates that, relative to PBN, nitrone 1(Nu=OEt) produces a roughly equal amount of the corresponding doublespin adduct. It should be noted that the one election oxidationpotential of 1 (Nu=OEt) is much lower (0.48-D.52V) than that of PBN.That double spin adduct 6 is not an artifact produced via a mechanisminvolving the intermediacy of carbanionic species is supported by theabsence of the compound (1-cyanocyclohexyl)-diphenylmethanol in thereaction mixture (as determined by ¹ H NMR and TLC comparison with anauthentic sample) when the thermolysis is conducted in the presence ofan equimolar concentration of benzophenone. In toluene solution,benzophenone is preferentially attacked by carbanions (e.g.,organolithium derivatives) in the presence of equimolar concentrationsof nitrone 1 (Nu=OEt).

Results concerning the use of 1 (Nu=OEt) in trapping other types ofcarbon-centered radicals (such as aryl radicals) have likewise beenencouraging. For example, on the basis of ¹ H NMR analysis of thespectra of the violet and green products formed when nitrone 1 (Nu=OEt,10 mg, 100 mM in benzene) is subjected to conditions for the generationof the 4-bromophenyl radical (according to the Gokel modification of theGomberg-Bachmann reaction), their structures have been assigned as thecorresponding hydroxylamine (violet) and nitrone (green) resulting fromdisproportionation of the expected intermediate nitroxide radical. Thatthese products are also formed in 9:1 benzene:t-BuOH argues stronglyagainst their being artifacts formed via the involvement of arylcarbanion intermediates. See, e.g., Beadle, J. R. et al., in J. Org.Chem. (1984) 49:1594.

5.2.3. Oxidation and Chromotropism

Chromotropism has also been observed in experiments employing nitronespin trap 1 (Nu=OEt), and like compounds, in lipid peroxidation studies.Dissolution of 10 mg of 1 (Nu=OEt) in 50 ml of corn oil and bubbling ofair through the resulting green oil at 90° C. for 9.5 hr yields a brightred oil from which, after partitioning between hexane and acetonitrile,one can isolate from the acetonitrile layer 300 mg of crude reddishmaterial, which when subjected to TLC analysis, shows the presence of amajor red product. This red product has been identified as aldehyde 4(See, Scheme I, above) and is postulated to arise from decomposition ofa spin adduct between nitrone 1 (Nu=OEt) and an oxygen-centered radical.Nitrone 1 (Nu=OEt, 5.65×10⁴ M) is unchanged in 98:1:1 EtOAc:HOAc:waterafter 10 hours at 90° C. in a sealed tube. The green nitrone 1, then, onreaction with a peroxide radical (e.g., hydroperoxide HOO·, oralkylperoxide ROO·) gives rise to a combination product that is violet(presumably, a nitroxide adduct), which on hydrolysis (or some otherdecomposition reaction, such as hyrogen abstraction, disproportionation,fragmentation and the like) gives rise to an end product that is red,corresponding to the aldehye 4 (1-(3-carboxylicacid-7-isopropyl-4-methyl)azulenyl carboxaldehyde, ethyl ester).

The difference in color between the nitrone and the aldehyde isreflected in the differences in the UV/VIS absorption spectra of the twocompounds in alcoholic solvent, in which the nitrone has a strongabsorption around 305 nm and the aldehyde a sharp peak around 255 nm.Both compounds have medium absorption peaks around 390 nm.

The electrochemical oxidation of the of the azulenyl nitrone into thealdehyde appears to take place around 600 mV in an aqueous environment.The redox reaction in acetonitrile is reversible, indicating anoxidation potential of 0.84 V v. SCE for the nitrone 1.

Further, when a control experiment is conducted with argon bubbling inthe presence or absence of water, this red product is not detected andthe recovered green oil contains no azulenic products other than thestarting azulenyl nitrone. Similarly, no observable chromotropism andcomplete recovery of the unreacted nitrone is the outcome of an aerobiccontrol experiment substituting chlorobenzene, a solvent devoid ofeasily abstractable hydrogen atoms, in place of corn oil. This datastrongly suggests that the observed change in color from green to red isinstigated by addition to nitrone 1 Nu=OEt) of free radicals formed byautoxidation, presumably involving linoleic acid subunits of the cornoil glycerides. The application of these nitrones as indicators ofoxidative stress in lipids is thus demonstrated.

Moreover, by virtue of the presence of acyl bromide intermediate 2 (See,Scheme I, above) in the synthesis of these nitrones, one can envisionmaking a wide range of easily prepared derivatives whose physicalproperties can be modulated by judicious choice of a nucleophile(alcohol, amine, etc.) to employ in acylation reactions with 2.Lipophilic or hydrophilic side chains at this position shoulddrastically influence solubility properties and the exploitation of thiselectrophilic site for the preparation of bioconjugates may affordinteresting spin traps with efficient targeting capacities.

The free radical scavenging and antioxidant properties of nitrones havebeen recent topics fostering intense activity in thebiological/pharmacological arena. Much evidence points to the role offree radical damage in the etiology of a number of pathologicalconditions such as atherosclerosis, Alzheimer disease, cancer,ischemia-reperfusion injury, and senescence.

5.2.4. Azulenyl Nitrones on Solid Supports

Since azulenyl nitrones exhibit chromotropism, these compounds areuseful as indicators of free radicals reagents in a gas, liquid, orsolid medium. In this example, the azulenynl nitrones are bound to asolid support (e.g., via reactive functional groups, such as hydroxylgroups, on the solid support). The solid support bound azulenyl nitroneis generally insoluble in the medium to be tested or monitored for thepresence of free radicals. Hence, this method of the detection of freeradicals can be performed without contaminating the medium. The azulenylnitrones are bound to a solid support, which may be in the form ofbeads, solid strip, paper, or any form suitable for the testingconditions. Furthermore, the reaction product generated between theazulenyl nitrone and any free radical can be isolated by simplyfiltering the solid support. The reaction product can then be isolatedfrom the solid support, free of the medium, by hydrolyzing or breakingthe bond binding the azulenyl nitrone (actually the spin adduct) to thesolid support. This method permits the identification of the freeradicals that are present in the medium being tested and which give riseto the formation of the spin adduct.

For example, polyvinyl acetate beads are partially hydrolyzed, therebyexposing hydroxyl groups on the surface capable of bonding to theazulenyl nitrone (e.g., the acid form). The amount of hydrolysis of thebeads and the concentration of azulenyl nitrone substituents are variedto adjust the sensitivity of the beads to free radicals. The beads areimmersed in vegetable oil. The mixture is heated and exposed to airbubbling through the mixture resulting in a visible color change. Thebeads are removed from the vegetable oil, thereby removing anycontaminant. The reaction product generated with the azulenyl nitroneand radical is isolated by hydrolyzing the product with a base. Theunbound reaction product can then be analyzed by conventional methods.

5.3. In Vitro and In Vivo Studies Using Azulenyl Nitrones

5.3.1. Protective Effect Against Oxidative Damage

To study the protective effects of azulenyl nitrones, such as 1(Nu=OEt), against oxidative damage induced by various modalities, theclonogenic cell survival is a reliable end point. A diverse group ofcells ranging from prokaryotes to mammalian cells can be used.Preferably, because of their rapid doubling time and high platingefficiency, Chinese hamster V79 lung fibroblasts are chosen, which aregrown in sterile Ham's F12 medium supplemented with 10% fetal calf serumwith glutamine and without sodium bicarbonate (Hyclone Laboratories,Logan, Utah), penicillin at 0.14 mg/ml, and streptomycin at 0.2 mg/ml.

Drug treatment or high-energy irradiation is performed in the presenceor absence of varying concentrations (0.1-100 mM) of the azulenylnitrones of the present invention. In a typical experiment, 5×10⁵ cellsin 5 ml medium are plated into a 100-mm petri dish and incubated (95%air/5% CO₂, by volume) for 16 hr at 37° C. Following cell adherence tothe plates and exponential growth, 10 mM 1 (Nu=OEt) and 0-1.2 mM H₂ O₂are added. After 1 hr the cells are rinsed, treated with 0.03% trypsin,counted, and divided into dishes to be incubated for macroscopic colonyformation. After 7 days the cells are fixed with methanol/acetic acid(3:1, v/v), stained with 0.3% crystal violet, rinsed, and air-dried, andthe colonies containing over 50 cells are counted. In this way thedose-dependent protective effect of azulenyl nitrones of the inventionare evaluated.

5.3.2. Brain Antioxidant Activity

The brain antioxidant activity of azulenyl nitrones are studied usingtwo groups of animals: (i) young adult male gerbils (3-4 months of age)and (ii) aged, retired, male breeder gerbils (18-20 months of age). Suchgerbils may obtained from Tumblebrook Farms (West Brookfield, Mass.).The gerbils are housed three in a cage in standard rodent cages. Animalsare maintained in an animal facility under a 12-hr light-dark cycle. Allexperiments are conducted during the light phase of the cycle. Food andwater are available ad lithium throughout the day.

Young adult male (3-4 months of age) and retired male breeder gerbils(18-20 months of age) are assigned to separate groups of 18 gerbilseach. One group of young adult and aged animals is assigned to thevehicle (saline) control group, and the other groups receive the sodiumsalt of Compound #4(Nu=OH). Animals are given intraperitoneal injectionstwice daily (8:00 a.m. and 8:00 p.m.) for a period of 14 consecutivedays. The sodium salt of Compound #4(Nu=OH) is dissolved in neutralsaline and administered at a dose of 1-50 mg, preferably 30mg/kg/injection (3.0 mg/ml). In subsequent studies, substantially lowerdoses (1-10 mg/kg/injection) are used with comparable results.

At the end of the 14 days, the animals are given an additional day of noinjections to allow for the elimination of any residual azulenyl nitroneprior to testing. After the 24 hour washout period, the gerbils aretested for radial arm maze performance.

An eight-arm, or like, radial maze is used for testing patrollingbehavior performance. The gerbils are placed one at a time in thecentral compartment of the sunburst maze. When the doors to the arms areraised, each gerbil is free to explore the maze. Reentry into an armmore than once before exploring all eight arms is considered an error.Arm entry is registered electronically. Animals have 15 min to explorethe maze.

Normal young adult gerbils make between 4 and 5 errors, while agedgerbils make 9-11 errors. After 14 days of treatment with the sodiumsalt of Compound #4(Nu=OH), the young adult gerbils make the same numberof errors as the control group In contrast, when nitrone-treated agedgerbils are tested they make significantly fewer errors. In fact, theaged gerbils surprisingly make about the same number of errors as do theyoung adult gerbils.

5.3.3. In Vivo Diagnostic Applications

Azulenyl nitrones are detectable by UV/VIS spectroscopy and HPLC. Uponreaction with free radicals and subsequent breakdown of the adduct, thenitrones generate the corresponding azulenyl aldehydes. Such aldehydesarise from the breakdown of spin adducts formed with oxygen-centeredfree radicals. The aldehydes are, in turn, also detectable by UV/VISspectroscopy. When a subject is dosed with nitrones and subsequentlytreated in some fashion (e.g., ischemia and/or reperfusion) to cause theformation of free radicals in some part of the subjects anatomy, theamount of free radicals induced in the subject can be gauged bymeasuring the amount of aldehyde produced. (The nitrone should berecovered unchanged if no oxidation reaction takes place.) Thus, thealdehyde to nitrone ratio can be determined in the subject's biologicalfluids and/or tissue samples (e.g., in the blood, cerebral, orcardiovascular tissue). In this fashion, azulenyl nitrones are used togauge the relative levels of reactive free radical production in variouslocations and/or fluids of the subject. Such analytical techniques canalso demonstrate that the azulenyl nitrones of the present invention areable to penetrate certain barriers that are present in the animal orhuman anatomy, such as the blood-brain barrier, or whether thesecompounds prefer to remain or localize in certain tissues (e.g.,hippocampus) or fluids (e.g., plasma) after different modes ofadministration, e.g., iv, ip, po, topical, intramucal, opthalmic, etc.

5.3.4. In Vivo Neuroprotective Applications

Because azulenyl nitrones react with free radicals, physiological eventsand/or pathological conditions that lead to the formation of freeradicals and which thereby cause damage to the subject suffering fromsuch event or condition can be prevented, inhibited, or alleviated bythe administration of azulenyl nitrones. A method of determining theeffectiveness of azulenyl nitrones as a neuro- or cerebroprotectantinvolves the administration of azulenyl nitrones to test animals,including rodents such as gerbils or mice. An ischemic episode is theninduced in the test animal. For example, a useful stroke model isprovided by subjecting the test animal to a bilateral carotid occlusion(BCO), which reduces the flow of blood to the brain and can result inbrain damage and/or tissue infarction. The blood and brains of theBCO-treated rodents are analyzed to determine the relative amounts ofazulenyl nitrone and aldehyde. The ratio of the aldehyde to nitroneconcentrations is compared to that found in sham rodents, which were notsubjected to one of the oxygen free radical producing procedure. Theresults indicate that the ratio is higher (i.e., that more aldehyde isobserved) in the test rodents versus the sham rodents, indicating thatthe administration of azulenyl nitrones to these rodents proceeds to aredox reaction/combination, which affords the end product, aldehyde.

The administration of the azulenyl nitrones of the invention to the testrodents affords neuroprotection because control animals receiving saline(or phenyl t-butyl nitrone or PBN) exhibit impaired motor functionand/or behavior compared to the test animals. Moreover, analysis ofbrain slices and heart slices indicates a reduction in the cerebral andmyocardial infarct volume, respectively, relative to saline control oranimals receiving PBN.

5.3.5. In Vitro Protection

The protective effects of azulenyl nitrones are investigated bysubjecting a cell culture to compounds known to induce death in thecells. The nitrone is administered in varying doses to determine theamount needed to inhibit or prevent cell death. By this method, theefficacy of azulenyl nitrones is determined and found to be largely dosedependent.

As an example, cerebella granular cells (neuronal cells) are treatedwith a sublethal dose of a toxic agent, e.g., cis-platin, buthioninesulfoximine, or peroxynitrite. The nitrone is either added prior to orafter treatment with the toxic agent. Azulenyl nitrone 1 is found to bea neuroprotectant in a dose dependent manner, preventing or reducingneural cell death in doses of between 10 to 100 μM.

Similarly, it is found that the azulenyl nitrones of the invention, whenadded to cell cultures, particularly prokaryotic, eukaryotic, andespecially mammalian cells, extend the period of cell viability relativeto a control cell culture that received no azulenyl nitrone. Hence, theinvention inhibits cell apoptosis. (See, e.g., Schulz, J. B. et al., inJ. Neuroscience (1996) 16:4696-4706.). Similar results are obtained witha variety of neuroprotective cell toxicity assays.

5.3.6. Protection of Liver Oxidation

The protective effect of azulenyl nitrones on the formation of oxidativedamage in liver DNA and on lipid peroxidation is demonstrated byexperiments using Long-Evans Cinnamon (LEC) rats. See,.e.g., Yamashita,T. et al., in Free Radical Bio. & Med. (1996) 21:755-761. These ratsbelong to a new mutant strain with hereditary hepatitis and are used asmodels for treating Wilson's disease. LEC rats die of fllninanthepatitis within about a week of the development of severe jaundicewithout intervention.

In the experiment, the rats are maintained under conventionalconditions. Food and water are available ad lithium throughout the day.Two sets of female rats are used between the ages of 10 to 30 monthsold. To one set is subcutaneously administered the azulenyl nitrone 34in a vegetable oil composition, while the second set is subcutaneouslyadministered vegetable oil alone. The amount administered corresponds toabout 100 mg/kg of active ingredient and is administered twice daily forabout 15 to 30 weeks. The liver tissue of the rats is removed andmeasured for lipid peroxidation according to the method of Uchiyama, M.et al., in Anal. Chem. (1978) 86:271-278. In this way the dose-dependentprotective effect of azulenyl nitrones of the invention are evaluatedand shown to be remarkable.

5.4. Pharmaceutical Compositions Comprising the Azulenyl NitroneCompounds of the Present Invention

As should be apparent, the present invention contemplates compositionscomprising the azulenyl nitrone compounds disclosed herein. Preferably,these compositions include pharmaceutical compositions comprising atherapeutically effective amount of an azulenyl nitrone compound alongwith a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically acceptable" carrier means anon-toxic, inert solid, semi-solid liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. Some examples of thematerials that can serve as pharmaceutically acceptable carriers aresugars, such as lactose, glucose and sucrose, starches such as cornstarch and potato starch, cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt, gelatin, talc; excipients such as cocoa butter andsuppository waxes; oils such as peanut oil, cottonseed oil, saffloweroil, sesame oil, olive oil, corn oil and soybean oil; glycols, such aspropylene glycol, polyols such as glycerin, sorbitol, mannitol andpolyethylene glycol; esters such as ethyl oleate and ethyl laurate,agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer'ssolution; ethyl alcohol and phosphate buffer solutions, as well as othernon-toxic compatible substances used in pharmaceutical formulations.Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfateand magnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and conventional antioxidants can also be present in thecomposition, according to the judgement of the formulator. Examples ofpharmaceutically acceptable conventional antioxidants include watersoluble antioxidants such as ascorbic acid, cysteine hydrochloride,sodium bisulfite, sodium metabisulfite, sodium sulfite, and the like;oil soluble antioxidants such as ascorbyl pahmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol and the like: and the metal chelating agentssuch as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid and the like.

By a "therapeutically effective amount" of an azulenyl nitrone compound,such as 1 (Nu=OEt) or the sodium salt of Compound #4(Nu=OH), is meant asufficient amount of the compound to alleviate, modulate, or inhibit thenegative or, otherwise, ill effects of free radical species and/orassociated medical disorders at a reasonable benefit/risk ratioapplicable to any medical treatment. It will be understood, however,that the total daily usage of the compounds and compositions of thepresent invention will be decided by the attending physician within thescope of sound medical judgement. The specific therapeutically effectivedose level for any particular patient will depend upon a variety offactors including the medical disorder being treated and the severity ofthe medical disorder; activity of the specific compound employed; thespecific composition employed; the age, body weight, general health, sexand diet of the patient; the time of administration, route ofadministration, and rate of excretion of the specific compound employed;the duration of the treatment; drugs used in combination or coincidentlywith the specific compound employed; and like factors well known in themedical arts.

The total daily dose of the azulenyl nitrone compounds of the presentinvention administered to a human subject in single or in divided dosescan be in amounts, for example, from 0.01 to 35 mg/kg body weight ormore usually from 0.1 to 15 mg/kg body weight. Single dose compositionsmay contain such amounts or submultiples thereof to make up the dailydose. In general, treatment regimens according to the present inventioncomprise administration to a human or other mammal in need of suchtreatment from about 1 mg to about 1000 mg of the compound(s) of thisinvention per day in multiple doses or in a single dose offrom 1 mg, 5mg, 10 mg, 100 mg, 500 mg or 1000 mg. A submilligram dose may also beappropriate, namely, about 0.1-0.9 mg, preferably, about 0.3, about 0.5,or about 0.7 mg.

The compounds of the present invention may be administered alone or incombination or in concurrent therapy with other agents that exhibitantioxidant activity, such as PBN.

Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs containing inert diluents commonly used in the art such aswater. Such compositions may also comprise adjuvants, such as wettingagents; emulsiying and suspending agents; sweetening, flavoring andperfuming agents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulation can be sterilized, for example, by filtrationthrough a bacteria-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedor dispersed in sterile water or other sterile injectable medium justprior to use.

In order to prolong the effect of a therapeutic agent, it is oftendesirable to slow the absorption of a therapeutic agent fromsubcutaneous or intramuscular injection. The most common way toaccomplish this is to inject a suspension of crystalline or amorphousmaterial with poor water solubility. The rate of absorption of thetherapeutic agent becomes dependent on the rate of dissolution of thetherapeutic agent which is, in turn, dependent on the physical state ofthe therapeutic agent, for example, the crystal size and the crystallineform. Another approach to delaying absorption of a therapeutic agent isto administer the therapeutic agent as a solution or suspension in oil.

Injectable depot forms can also be made by forming rnicrocapsulematrices of therapeutic agent and biodegradable polymers such aspolylactide-polyglycoside. Depending on the ratio of therapeutic agentto polymer and the composition of the polymer, the rate of therapeuticagent release can be controlled. Examples of other biodegradablepolymers include poly-orthoesters and polyanhydrides. The depotinjectables can also be made by entrapping the therapeutic agent inliposomes or microemulsions which are compatible with body tissues.

Suppositories for rectal administration of the therapeutic agent can beprepared by mixing the therapeutic agent with a suitable nonirritatingexcipient such as cocoa butter and polyethylene glycol which are solidat ordinary temperature but liquid at the rectal temperature and will,therefore, melt in the rectum and release the therapeutic agent.

Solid dosage forms for oral administration may include capsules,tablets, pills, powders, prills and granules. In such solid dosage formsthe active spin trapping compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such asmagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. Tablets and pills can additionally be prepared withenteric coatings and other release-controlling coatings.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The active nitrone compounds can also be in micro-encapsulated form withone or more excipients as noted above. The solid dosage forms oftablets, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings and other coatings well known in thepharmaceutical formulating art. They may optionally contain opacifyingagents and can also be of a composition that they release the activeingredient(s) only, or preferably, in a certain part of the intestinaltract, optionally in a delayed manner. Examples of embeddingcompositions which can be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a nitronecompound of this invention, for either therapeutic or cosmeticapplications, further include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants (e.g., through the oral cavity orintranasally) or patches. The active nitrone component is admixed understerile conditions with a pharmaceutically acceptable carrier and anyneeded preservatives or buffers as may be required. Ophthalmicformulations, ear drops, eye ointments, powders and solutions are alsocontemplated as being within the scope of this invention.

The ointments, pastes, creams and gels may contain, in addition to anactive nitrone compound of this invention, excipients such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the compounds of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispersing the compound in the proper medium. Absorptionenhancers can also be used to increase the flux of the compound acrossthe skin. The rate can be controlled by either providing a ratecontrolling membrane or by dispersing the compound in a polymer matrixor gel.

Accordingly, the present invention is useful in the treatment oralleviation of disease, especially those disorders related to oxidizedspecies, free radicals, or products of oxidation, including products ofpolymorphonuclear leukocyte oxidative burst. Such medical conditions maybe characterized by inflammation, rheumatoid arthritis, autoimmunedisease, flu-like symptoms, decreased cognitive ability, cardiovasculardisease, atherosclerosis, respiratory discomfort and the like, which canbe reduced by the administration of an effective amount of the azulenylnitrone compounds of the present invention.

Reactive free radicals in living tissue are believed to promote heartdisease, cancer, Alzheimer's disease, Parkinson's disease, ainyotrophiclateral sclerosis (or ALS), rheumatoid arthritis and even antineoplastic(anticancer, antitumor) induced cardiotoxicity. There exist manymechanisms that induce the formation of free radicals in livingorganisms. Some occur naturally, such as through the metabolic process,while others are introduced into the body by way of chemical agents,radiation, microbes and viruses.

The initial presence of the free radical initiates a chain reaction inwhich a number of biomolecules in the organism are oxidized. Byoxidizing lipids, for example, these free radicals can affect cellmembranes, the permeability of cell membranes, ion channels containedtherein, cell function, etc. By oxidizing proteins, for example, freeradicals can alter enzymes, muscular function, nerves, etc. And byoxidizing nucleic acids, for example, free radicals can affect DNA, RNA,and consequently their function, regulation, or expression products.Spin trapping agents are utilized to terminate or inhibit this damagingcascade of reactions. It has been found that oxygen-centered freeradicals and carbon-, nitrogen-, phosphorous- and sulfur-centeredradicals react more readily with the spin trapping agent of theinvention than with the potential target biomolecules. The reaction withthe spin trapping agent results in the formation of a stable spin adductand thus, terminates and/or inhibits the damaging chain reaction.

Hence, the azulenyl nitrone compounds of the present invention can beused in a method of treating, alleviating, modulating, or inhibiting theeffects in the heart or brain of ischemia or reperfusion injury, acuterespiratory distress syndrome (ARDS), sepsis, septic shock and the like.The invention also demonstrates a capacity to preserve organs prior totransplantation comprising contacting the organ to be preserved with anorgan preserving effective amount of a compound of the invention.

The phrase "pharmaceutically acceptable salt" includes any type of saltof the azulenyl nitrones of the present invention, whether derived fromthe addition to the nitrone of a base or an acid, which is suitable forpharmacologic use. Hence, the salt can be obtained by the addition of aalkali or alkaline earth substance (e.g., sodium hydroxide, calciumcarbonate, magnesium sulfate and the like) to a nitrone bearing anacidic group (e.g., carboxylic acid or sulfonic acid). Conversely, anyfree basic functional groups (such as an amino group) on the nitrone canbe treated with an acidic substance (e.g., hydrochloric acid, nitricacid and the like) to provide an acid addition salt.

The compounds of the invention can be administered alone or incombination with one or more other biologically active (preferably,therapeutically active) agents, either substantially simultaneously orsequentially. An effective amount of azulenyl nitrone, co-administeredwith a second agent exhibiting some tissue necrosis or toxicity, mayreduce the harmful side effect of the co-administered drug while stillderiving the benefit of the therapeutic effect of the second drug.Hence, a combination comprising a therapeutically effective amount ofadriamycin, taxol, cis-platin, or other anticancer agents, or AZT, DDI,or other protease inhibitors and an amount of azulenyl nitrone effectiveto reduce toxicity associated with the other drug(s) is expresslycontemplated.

5.5. Other Specific Embodiments and Illustrative Methods

The present invention further contemplates compounds of the formula:##STR6## in which R₁ may be a hydrogen, a linear or branched alkyl groupcomprising 1-6 carbon atoms, or an aryl group comprising 6-10 carbonatoms; R₂ may be a linear or branched alkyl group comprising 1-6 carbonatoms, or an aryl group comprising 6-10 carbon atoms; R' may be a linearor branched alkyl group comprising 1-6 carbon atoms; W may be a linearor branched alkyl group comprising 1-6 carbon atoms, an aryl groupcomprising 6-10 carbon atoms, or an electron-withdrawing group; m may be0, 1, 2, or 3 (if m is 2 or 3, each R' may be the same as or differentfrom one another) or a salt thereof. Specific compounds may, of course,be in the form of its metal salt, such as an alkali or alkaline-earthmetal salt, its ammonium or tetraalkylammonium salt. A preferredazulenyl nitrone appears green to the naked eye.

In addition, the invention provides a method of trapping a reactive freeradical comprising providing a compound of the general formula: ##STR7##as already defined, above; and allowing the compound to combine with areactive free radical to provide a spin adduct comprising a combinationproduct of the compound or its salt and a free radical.

In specific embodiments of the invention, the free radical iscarbon-centered or is centered on a heteroatom. In particular, theheteroatom is selected from nitrogen, oxygen, phosphorus, or sulfur. Thefree radical can also be centered on a metal, especially a heavy metal,or more particularly, a transition metal, an actinide metal, or alanthanide metal. Specific free radicals, which are contemplated to forman adduct with an azulenyl nitrone of the invention include, but are notlimited to, singlet oxygen, hydroxyl, superoxide, hydroperoxide,alkylperoxide, or nitric oxide radical. The free radical may alsoderived from a photosensitizer.

The adduct can undergo further reactions to generate compounds of theformula: ##STR8## in which R1, R2, R3, R4, R', W, p, n, o and m are asdefined previously and X is oxygen, nitrogen, or sulfur.

Yet other methods include a method of detecting oxidation products in amedium comprising combining a compound of the general formula, above, orits salt with a medium and detecting the presence of an adduct or anend-product thereof. Such methods may further comprise the structuralcharacterization of the spin adduct formed or an end-product thereof toobtain information relating to the initial free radical or oxidativespecies. In the methods, above, the medium may be any solid, liquid, orgaseous medium, but preferably one that comprises a combustible fuel,lubricant, solvent, foodstuff (e.g., meat, poultry, fish, frying oil,vegetable oil), or a biological fluid (e.g., whole blood, peripheralblood, plasma, serum, cerebrospinal fluid, urine, semen, tears, saliva,mucus and the like) or a fraction thereof. The medium may also be a cellculture or a supernatant thereof.

In yet another method, azulenyl nitrone compounds can be used in thescreening of natural products that readily give rise to free radicals,e.g., enediyne antibiotics, such as bleomycin, or iron-centered drugs,which may eventually bind DNA/RNA.

Specific compositions include, but are not limited to, a pharmaceuticalcomposition for alleviating a the ill effects of a pathologic conditionmediated or initiated by a reactive free radical, in which thecomposition comprises an effective amount of the compound of the generalformula and a pharmaceutically acceptable carrier. Other compositionscomprising the compounds of the present invention and a carrier are alsocontemplated including, but not limited to, those that inhibitoxidation, a fuel additive, a food additive (such as one that is addedto a vegetable oil), a cosmetic (such as a facial or body sunscreen ofcharacteristic colors and which change color, indicating overexposure tooxidative conditions or elements). Still other compositions may be thosethat alleviate the ill effects of aging and in which the carrier issterile.

Still another objective of the invention is a process for malking anazulenyl nitrone comprising: (a) providing an azulene: (b) introducing aacyl group to the azulene at a position that is to bear a nitrone group;(c) converting the acyl group to a nitrone group to provide an azulenylnitrone. The process contemplated could further comprise introducing asecond acyl group to the azulene at a position that is to bear a groupdesignated W, or it could further comprise converting the second acylgroup to a group designated W. Preferably, the group designated Wcomprises an electron-withdrawing group and that the acyl groupcomprises an aldehyde. The second acyl group may comprise an acylhalide. Specifically, the group designated W comprises a carboxylicacid, its ester, amide, or salt.

Generally, thus, those additional applications of the present inventionlead to a method of alleviating the ill effects of ischemia orreperfusion injury in a subject comprising administering to the subjectan effective amount of a compound of the invention, a method ofalleviating the ill effects of Acute Respiratory Distress Syndrome(ARDS) in a subject comprising administering to the subject an effectiveamount of a compound of the invention, or a method of alleviating theill effects of aging, apoptosis, or senescence in a subject comprisingadministering to the subject an effective amount of a compound of theinvention.

The present invention also contemplates a composition for the treatmentof an inflammation in a warm-blooded animal comprising an azulenylnitrone of the invention a topical carrier. The composition of may comein the form of an aqueous solution, oil, cream, cake, powder, emulsion,or suspension. Moreover, the nitrone may further comprise a group W thatis an unsaturated aliphatic group comprising 2-14 carbon atoms. Theunsaturated aliphatic group can be further substituted by anelectron-withdrawing group, an aryl group comprising 6-18 carbon atoms,a saturated or unsaturated monocyclic or polycyclic ring systemcomprising 5-20 carbon atoms. Alternatively, the unsaturated aliphaticgroup may include a substituted or unsubstituted azulene. The group Wmay simply includes a hydrophilic moiety, such as a beta-lactam. Inparticular, the group W may include a 2-pyrrolidone group or is acarboxylic acid, 2-(2-pyrrolidon-N-yl)ethyl ester.

Yet another compound of the invention has the general formula ##STR9##in which R₁ may be a hydrogen, a linear or branched alkyl groupcomprising 1-6 carbon atoms, or an aryl group comprising 6-10 carbonatoms; R₂ may be a linear or branched alkyl group comprising 1-6 carbonatoms, or an aryl group comprising 6-10 carbon atoms; R₃ may be ahydrogen, or a linear or branched alkyl group comprising 1-6 carbonatoms; R₄ may be a hydrogen, or a linear or branched alkyl groupcomprising 1-6 carbon atoms; R₅ may be a hydrogen, or a linear orbranched alkyl group comprising 1-6 carbon atoms; R₆ may be a hydrogen,or a linear or branched alkyl group comprising 1-6 carbon atoms; R' maybe a linear or branched alkyl group comprising 1-6 carbon atoms; W maybe a linear or branched alkyl group comprising 1-6 carbon atoms, an arylgroup comprising 6-10 carbon atoms, or an electron-withdrawing group; nmay be 0, 1, or 2 (if n is 2, each W may be the same as or differentfrom one another); m may be 0, 1, 2, or 3 (if is 2 or 3, each R' may bethe same as or different from one another); o may be 1 or 2 (if o is 2,each R₁ and R₂ may be the same as or different from one another); p maybe 0, 1, or 2 (if p is 2, each R₃ and R₄ may be the same as or differentfrom one another); q may be 0, 1, 2, 3 or 4 (if q is greater than 1, R₅and R₆ may be the same as or different from one another) or a saltthereof.

It is hoped that the invention has been described herein in a mannerthat allows one of ordinary skill ample and adequate disclosure topractice the invention. In an overabundance of caution, however, thefollowing detailed examples are provided further consideration of theinterested reader.

6. EXAMPLES

6.1. Preparation of Azulenyl Nitrones

6.1.1. Materials and Methods

A number of azulene starting materials are known or commerciallyavailable. Azilene, for example, is available from Aldrich Chemical Co.(Cat. No. A9,720-3). Guaiazulene, 7-isopropyl-1,4-dimnethylazulene, isalso sold by Aldrich (Cat. No. G1-100-4). Guaiazulene can also beisolated from chamomile oil or guaiac wood oil. Its 3-sulfonic acid,sodium salt, derivative is known as an anti-inflammatory andanti-ulcerative agent. The total synthesis of guaiazulene is describedby Plattner et al., in Helv. Chim. Acta (1949) 32:2452. Thepharmacokinetics of guaiazulene 3-sulfonate sodium salt in animals isdescribed by Mukai, H. et al., in J. Pharmacobio-Dyn. (1985) 8:329, 337.The effect of guaiazulene or its salt on gastric and duodenal ulcers inrat models has been described. See, Okabe, S. et al., in NipponYakurigaku Zasshi (1986) 88:467; Chem. Abstr. (1987) 106:43769.

4,6,8-Trimethylazulene can be purchased from Fluka. Lactarviolin,7-isopropenyl-4-methyl-1-azulenecarboxaldehyde, is an antibiotic pigmentproduced by the fungus Lactarius deliciosus. Chamazulene,7-ethyl-1,4-dinethylazulene, is an anti-inflammatory agent that can beobtained from chamazulenogenic compounds found in chamomile, wormwoodand yarrow. Chamazulene is a blue oil, but its trinitrobenzenederivative, mp 131.5-132.5 degrees C., provides dark violet needles fromabsolute ethanol. Other potential starting materials include4-methyl-1-azulenecarboxaldehyde, a liverwort component andlinderazulene, a tricyclic 1,4-dimethylazulene derivative containing a3'-methylfuranyl ring fused to the 7- and 8-positions (the furan oxygenis attached to the 8-position) of the azulene nucleus. Other potentialazulene starting materials are known to those of ordinary skill in theart.

Melting points were determined on a Thomas-Hoover Meltemp apparatus andare uncorrected except where indicated. ¹ H NMR spectra were recorded ona General Electric 300-MHz instrument. Chemical shifts are reported invalues (parts per million, ppm) relative to an internal standard oftetramethylsilane in CDCl₃, except where noted. Abbreviations used inNMR analysis are as follows: s, singlet; d, doublet; t, triplet; m,multiplet; dt, doublet of triplets. Analytical thin-layer chromatography(TLC) was performed on Baker-flex silica gel 1B2-F plastic plates.Microanalyses were obtained from the Florida International UniversityMicroanalytical Laboratory and from Galbraith Laboratories, Inc.Solvents and reagents were used as purchased, except as noted. TEF wasdistilled from sodium metal/benzophenone ketyl.

6.1.2. General Procedure A

Beginning with a starting azulene, such as guaiazulene, anelectron-withdrawing substituent (e.g., a carboxylic acid ester) can beplaced on the ring system by the following procedure: To a 0.1M solutionof starting azulene in dry Et₂ O at rt is added oxalyl bromide (1.0 eq)dropwise over 15 minutes under argon with stirring. The mixture isstirred at rt for 1 hour and then 2 eq of EtOH is added dropwise over 10minutes. The resulting mixture is stirred an additional hour at rt andis then poured into a separatory final containing Et₂ O and sat aq.NaHCO₃ solution. The Et₂ O layer is washed with H₂ O, dried over MgSO₄and evaporated to provide the desired product in 80% yield. It isimportant to note that virtually any nucleophile, other than the EtO⁻illustrated here, can be introduced to the carbonyl-containingelectron-withdrawing group by allowing the desired nucleophile to reactwith the acyl bromide intermediate.

6.1.3. Procedure B

A 1-methyl substituent can be oxidized to a carboxaldehyde group by thefollowing procedure: To a stirred mixture of 100 ml of acetonitrile, 5ml of water and 3.7 mmol of the azulenyl ester of Procedure A at roomtemperature is added 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (7.8mmol) in one portion. The reaction mixture is stirred at rt for 60minutes. The contents of the reaction flask are then poured into 2liters of CHCl₃ and the solution is dried over MgSO₄, filtered andconcentrated to give a brown solid. This solid is purified by columnchromatography on silica gel (elution with CHCl₃) to give a red solid.Dissolution of this solid in 250 ml of Et₂ O and treatment with 150 mlof sat. aq. sodium thiosulfate solution in a separatory funnel provides,after vigorous shaking, a red ether layer that is subsequently washedwith 100 ml of sat. aq. sodium chloride solution. The washed organicsolution is dried over anhydrous magnesium sulfate, filtered andconcentrated to furnish 2.74 mmol (74%) of the solid red aldehyde.

6.1.4. Procedure C

A carboxaldehdye group is converted readily to, for example, aN-tert-butyl nitrone group, by the following method: To a 0.2M solutionof the aldehyde obtained by Procedure B in dry pyridine under argon atrt is added solid N-tert-butylhydroxylamine hydrochloride (1.5-2.0 eq).The mixture is heated with stirring to 95° C. for one hour and thenallowed to cool to rt. The pyridine is removed on the rotary evaporator,and the residue is dissolved in CHCl₃. The CHCl₃ layer is washed withwater and dried over MgSO₄. Evaporation of the CHCl₃ followed by silicagel chromatography of the residue (CHCl₃ :MeOH) provides the solid greenazulenyl nitrone in 96% yield.

6.1.5. Procedure D

A carboxaldehyde group can be introduced, e.g., at the 1-position ofazulene by treating the azulene starting material with POCl₃ in DMF (theVilsmeier reaction). See, Hafner, K. and Bernhard, C., in Angew. Chem.(1957) 69:533; Treibs et al. Chem. Ber. (1959) 92:141. The resultingaldehyde can then be converted to the nitrone by the method of ProcedureC.

6.1.6. Procedure E

Likewise, a sulfonate group can be introduced, e.g., at the 3-position,of guaiazulene by treating the starting azulene with SO₃ in dioxane,followed by treatment of the resulting sulfonic acid with a base, suchas sodium hydroxide. See, Miyazaki, S. et al., in Japanese Patent Publ.No. 3065; Chem. Abstr. (1960) 54:13090.

6.1.7. Procedure F

Two carboxaldehdye groups are converted readily to, for example,N-tert-butyl nitrone groups, by the following method: To a 0.5M solutionof the di-aldehyde in dry pyridine under nitrogen at rt is added solidmagnesium sulfate (0.1-0.4 eq) and solid N-tert-butylhydroxylaminehydrochloride (2.0-4.0 eq). The mixture is heated with stirring to 95°C. and is stirred overnight. Upon cooling to rt, the reaction mixture ispoured into a separator funnel containing 60 ml of CHCl₃ and 60 ml ofsat aq. NaHCO₃. The aqueous layer is separated and washed with three 30ml portions of CHCl₃. The combined organic layers are dried overanhydrous MgSO₄, filtered, and evaporated to give the pure desiredbis-nitrone.

6.2. Syntheses of Representative Azulenyl Nitrones

Using one or more of the starting materials described above, or anyother azulene ring system of interest, numerous azulenyl nitrones can beprepared. Thus, for example, 2,4,6-trimethylazulene can be treated withoxalyl bromide in ether by the method of Procedure A to provide, afterthe addition of a suitable nucleophile, Nu⁻, a 3-CONu-substituted4,6,8-trimethylazulene. Subjecting this intermediate to the conditionsof the Vilsmeier reaction (Procedure B), followed by treatment of theresulting carboxaldehyde with N-tert-butylhydroxylamine hydrochloride inpyridine (Procedure C) provides the compound illustrated below:##STR10## in which the group Nu may be virtually any nucleophilic group,but preferably, hydroxyl, lower alkoxy (e.g., methoxy, ethoxy and thelike), N,N'-di(lower alkyl)amide (e.g., diimethylamide, diethylamide andthe like), oxo salt, trifluoromethyl, spermine, N-methylglutamine, longchain aliphatic amine (e.g., C₈ -C₂₂ amino), sphingosine, polylysine, anantisense oligonucleotide sequence, a monoclonal antibody (preferablylinked via a connecting chain), a DNA intercalator (e.g., an acridineand the like), or a histone.

By following the procedures outline, above, the compounds listed in theTable, below, are prepared.

                                      TABLE                                       __________________________________________________________________________    Representative Azulenyl Nitrones                                               ##STR11##                                                                    Compound #                                                                           A           B  C             D       E F       G     H'                __________________________________________________________________________    1      CH═N.sup.+ (.sup.t Bu)O.sup.-                                                         H  CO.sub.2 Et   CH.sub.3                                                                              H H       .sup.i Pr                                                                           H                 2      CH═N.sup.+ (.sup.t Bu)O.sup.-                                                         H  CONME.sub.2   CH.sub.3                                                                              H H       .sup.i Pr                                                                           H                 3      CH═N.sup.+ (.sup.t Bu)O.sup.-                                                         H  CHO           CH.sub.3                                                                              H H       .sup.i Pr                                                                           H                 4      CH═N.sup.+ (.sup.t Bu)O.sup.-                                                         H  CO.sub.2 .sup.- Na.sup.+                                                                    CH.sub.3                                                                              H H       .sup.i Pr                                                                           H                 5      CH═N.sup.+ (.sup.t Bu)O.sup.-                                                         H  COCF.sub.3    CH.sub.3                                                                              H H       CMe═CH.sub.2                                                                    H                 6      CH═N.sup.+ (.sup.t Bu)O.sup.-                                                         H  CO-spermine   CH.sub.3                                                                              H H       .sup.i Pr                                                                           H                 7      CH═N.sup.+ (.sup.t Bu)O.sup.-                                                         H  CONEt.sub.2   CH.sub.3                                                                              H H       .sup.i Pr                                                                           H                 8      CH═N.sup.+ (.sup.t Bu)O.sup.-                                                         H  CON(Me)CH.sub.2 (CHOH).sub.4 CH.sub.2 OH                                                    CH.sub.3                                                                              H H       .sup.i Pr                                                                           H                 9      CH═N.sup.+ (.sup.t Bu)O.sup.-                                                         H  CONH(CH.sub.2).sub.17 Me                                                                    CH.sub.3                                                                              H H       .sup.i Pr                                                                           H                 10     CH═N.sup.+ (.sup.t Bu)O.sup.-                                                         H  CONH-sphingosine                                                                            CH.sub.3                                                                              H H       .sup.i Pr                                                                           H                 11     CH═N.sup.+ (.sup.t Bu)O.sup.-                                                         H  CONH-polylysine                                                                             CH.sub.3                                                                              H H       .sup.i Pr                                                                           H                 12     CH═N.sup.+ (.sup.t Bu)O.sup.-                                                         H  CONH-antisense                                                                              CH.sub.3                                                                              H H       .sup.i Pr                                                                           H                 13     CH═N.sup.+ (.sup.t Bu)O.sup.-                                                         H  CONH-mAb      CH.sub.3                                                                              H H       .sup.i Pr                                                                           H                 14     CH═N.sup.+ (.sup.t Bu)O.sup.-                                                         H  CONH-acridine CH.sub.3                                                                              H H       .sup.i Pr                                                                           H                 15     CH═N.sup.+ (.sup.t Bu)O.sup.-                                                         H  CONH-histone  CH.sub.3                                                                              H H       .sup.i Pr                                                                           H                 16     CH═N.sup.+ (.sup.t Bu)O.sup.-                                                         H  CH═N.sup.+ (.sup.t Bu)O.sup.-                                                           CH.sub.3                                                                              H H       .sup.i Pr                                                                           H                 17     CH═N.sup.+ (.sup.t Bu)O.sup.-                                                         H  H             CH.sub.3                                                                              H H       CMe═CH.sub.2                                                                    H                 18     CH═N.sup.+ (.sup.t Bu)O.sup.-                                                         H  H             H       H H       H     H                 19     H           H  H             H       H CH═N.sup.+ (.sup.t                                                        Bu)O.sup.-                                                                            H     H                 20     H           H  H             CH═N.sup.+ (.sup.t Bu)O.sup.-                                                     H H       H     H                 21     CH.sub.3    H  H             CH═N.sup.+ (.sup.t Bu)O.sup.-                                                     H H       .sup.i Pr                                                                           H                 22     CH.sub.3    H  H             CH.sub.3                                                                              H CH═N.sup.+ (.sup.t                                                        Bu)O.sup.-                                                                            .sup.i Pr                                                                           H                 23     CPh═N.sup.+ (.sup.t Bu)O.sup.-                                                        H  CO.sub.2 Et   CH.sub.3                                                                              H H       .sup.i Pr                                                                           H                 24     CH═N.sup.+ (.sup.t Bu)O.sup.-                                                         H  CN            CH.sub.3                                                                              H H       .sup.i Pr                                                                           H                 25     CH═N.sup.+ (.sup.t Bu)O.sup.-                                                         H  SO.sub.2 CH.sub.3                                                                           CH.sub.3                                                                              H H       .sup.i Pr                                                                           H                 26     CH═N.sup.+ (.sup.t Bu)O.sup.-                                                         H  SO.sub.3 .sup.- Na.sup.+                                                                    CH.sub.3                                                                              H H       .sup.i Pr                                                                           H                 27     CH═N.sup.+ (.sup.t Bu)O.sup.-                                                         H  P(O)(OMe).sub.2                                                                             CH.sub.3                                                                              H H       .sup.i Pr                                                                           H                 28     CH═N.sup.+ (.sup.t Bu)O.sup.-                                                         H  P(O)(O.sup.- Na.sup.+).sub.2                                                                CH.sub.3                                                                              H H       .sup.i Pr                                                                           H                 29     CH═N.sup.+ (.sup.t Bu)O.sup.-                                                         H  NO.sub.2      CH.sub.3                                                                              H H       .sup.i Pr                                                                           H                 30     CH═CHCH═N.sup.+ (.sup.t Bu)O.sup.-                                                H  CO.sub.2 Et   CH.sub.3                                                                              H H       .sup.i Pr                                                                           H                 31     CH═N.sup.+ (.sup.t Bu)O.sup.-                                                         H  H             CH.sub.3                                                                              H H       Ac    H                 32     CH═N.sup.+ (Ph)O.sup.-                                                                H  CO.sub.2 Et   CH.sub.3                                                                              H H       .sup.i Pr                                                                           H                 33     CH═N.sup.+ (.sup.t Bu)O.sup.-                                                         H  H             CH.sub.3                                                                              H H       .sup.i Pr                                                                           H                 34     CH═N.sup.+ (.sup.t Bu)O.sup.-                                                         H  CH═N.sup.+ (.sup.t Bu)O.sup.-                                                           H       H H       H     H                 __________________________________________________________________________

In particular, the following synthetic steps may be employed:

Compound #1 Starting with guaiazulene, use Procedure A, then B, then C.

Compound #2 Procedure A (substitute 2 eq. of HNMe₂ for EtOH), then B,then C.

Compound #3 Vilsmeier formulation of guaiazulene according to ProcedureD, then B, then C.

Compound #4 Procedure A (substitute NaOH for EtOH), then B, then C.

Compound #5 Protect aldehyde of lactaroviolin as dimethyl acetal(benzene, MeOH, cat. para-toluenesulfonic acid), then trifuoroacetylateWith (F₃ CCO)₂ O, ether, rt [Ref: Anderson, A. J., Jr. et al., in J.Org. Chem. (1965) 30:131], H₃ O⁺ hydrolysis of acetal, then C.

Compound #6 Procedure A (substitute 2 eq. [NB: 2d equiv scavenges HCl]spermine for EtOH), then B, then C [NB: reaction proceeds on 1° aminogroup of spermine].

Compound #7 Procedure A (substitute 2 eq. HNEt₂ for EtOH), then B, thenC.

Compound #8 Procedure A (substitute 2 eq. N-Methyl glucamine for EtOH),then B, the C.

Compound #9 Procedure A (substitute 2 eq. octadecyl-amine for EtOH),then B, then C.

Compound #10 Procedure A (substitute 2 eq. sphingosine for EtOH), the B,then C.

Compound #11 Procedure A (substitute polylysine for EtOH), then B, thenC.

Compound #12 Procedure A (substitute an antisense oligonucleotide, likepoly(dG)₁₀, for EtOH), then B, then C.

Compound #13 Procedure A (substitute a monoclonal antibody [NB: anymonoclonal Ab listed in ATCC Catalog,, for instance] for EtOH), then B,then C.

Compound #14 Procedure A (substitute H₂ N(CH₂)₃ NH[acridine] for EtOH),then B, then C [Ref: Plouvier, B. et al., in Bioconjugate Chemistry(1994) 5:475 (acridine bioconjugates)].

Compound #15 Procedure A (substitute a histone [NB: e.g., histonetype-II AS from calf thymus, available from Sigma] for EtOH), then B,then C.

Compound #16 Vilsmeier formylation of guaiazulene according to ProcedureD, then C with ≧2 eq. tert-butylNHOH·HCl.

Compound #17 Procedure C starting with lactaroviolin.

Compound #18 Procedure C with ≧2 eq. tert-butylNHOH·HCl starting with1-azulenecarboxaldehyde [NB: this starting material is obtained readilyby a Vilsmeier reaction involving azulene, according to Procedure D].

Compound #19 Starting with 6-azulenecarboxaldehyde [Ref. Huenig, S. etal., in Liebigs Ann. Chem. (1986) 1222 (synthesis6-azulenecarboxaldehyde and 4-azulenecarboxaldehyde)], obtain nitrone byProcedure C.

Compound #20 Starting with 4-azulenecarboxaldehyde, obtain nitrone byProcedure C.

Compound #21 Metalate guaiazulene by deprotonation at C-4 thyl groupusing NaNCH₃ Ph [Ref: Kurokawa, S., in Bull. Chem. Soc. Jpn. (1979) 1748(metalation of guaiazulene at C-4 methyl group)], then quench resultingorganosodium species with Cl(PO)(OMe)₂, metalate resulting phosphonatewith LDA/THF and perform Homer Wadsworth Emmons olefination withacetone, subsequent ozonolysis of double bond to give aldehyde, then C.

Compound #22 Same as for Compound #19 substituting guaiazulene forazulene.

Compound #23 This compound is produced by disproportionation of thenitroxide formed when Compound #1 captures a phenyl radical underconditions of the Gokel-modified Gomberg-Bachmann reaction: Compound #1(100 mM in benzene) is exposed to 1 eq. of Ph-N₂ BF₄, 2 eq. KOAc, and 5mol%, 18-crown-6 at rt for 1.5 hours [Ref: Gokel, G. W. et al., in J.Org. Chem. (1984) 49:1594]. Workup involves evaporation solvent andpurification by prep TLC (1:1 EtoAc:Hex). Compound #23 is green.

Compound #24 Cyanation of guaiazulene [Ref: Kitahara, Y. and Kato, T.,in Bull. Chem. Soc. Jpn. (1964) 37:859], then B, then C.

Compound #25 Treat guaiazulene with AlCl₃, ClSO₂ CH₃, CH₂ Cl₂, rt [Ref:Repogle, L. L. et al., in J. Org. Chem. (1967) 21:1909(3-azulenylsulfones)], then B, then C.

Compound #26 Sulfonation of guaiazulene according to Procedure E, thenB, then A.

Compound #27 Treat guaiazulene with AlCl₃, Cl(PO)(OMe)₂, CH₂ Cl₂, rt,then B, then C.

Compound #28 Same as Compound #27 except hydrolyze with NaOH beforesubjecting to Procedure B.

Compound #29 Catalytic hydrogenation of lactaroviolin, then nitrationwith HNO₃ /H₂ SO₄ at 0° C. in AcOH, then Procedure C.

Compound #30 Wittig reaction of Compound #1 with Ph₃ P=CHCHO, thenProcedure C.

Compound #31 Ozonolysis of lactaroviolin, then Procedure C.

Compound #32 Same as for Compound #1 substituting PhNHOH·HCl fortert-butylNHOH·HCl in Procedure C.

Compound #33 Catalytic hydrogenation of lactaroviolin, then Procedure C.

Compound #34 Starting with 1,3-Azulenedicarboxaldehyde [Ref: Hafner, K.and Bernhard, C., Annalen (1959) 625:108] obtain bis-nitrone byProcedure F.

Compound #35 Starting with 1,2-bis(guaiazulenylethylene, use ProcedureA, then B, then C.

6.3. Detection, Quenching and Characterization of Paramagnetic andDiamagnetic Species

6.3.1. Electron Spin Resonance Spectroscopy

The chromogenic azulenyl nitrones of the present invention facilitatethe detection of free radical species by providing a calorimetricindication of adduct formation. For example, the azulenyl nitrone ofCompound #1 is green, whereas the diamagnetic and paramagnetic spinadducts with carbon-centered radicals are violet. Paramagnetic speciesare detectable by ESR, as well. Isolation of the spin adduct can befacilitated by viewing colored bands chromatographic plates indicatingthe position of each chromophore, even when the chromophore is adiamagnetic combination, disproportionation, or reduction product of theinitially formed nitroxides.

Compound #1 (100 mM in benzene) is exposed to 1 equiv of the azocompound, (CH₃)₂ CHCH₂ (CH₃)C(CN)N═N(CN)C(CH₃)CH₂ CH(CH₃)₂, AMVN, andheated to 75° C. for 20 minutes. After cooling to room temperature, thesolution is transferred to an ESR tube, and examined by ESRspectroscopy. The ESR spectrum observed is shown FIGS. 1A and 1B. Thecontents of the ESR tube are applied to a prep TLC plate (SiO₂) andeluted with 99:1 (v/v) CHCl₃ :MeOH. The violet band is scraped from theplate and the SiO₂ extracted with 99:1 (v/v) CHCl₃ :MeOH. The solutionis evaporated to dryness and the residue is dissolved in 100 μl ofbenzene and transferred to a clean ESR tube. The ESR spectrum observedis identical to that recorded previously, indicating that the isolatedviolet product is responsible for the prior ESR signal.

Similarly, chromatographic separation of the highly colored diamagneticspin adducts can also be accomplished.

6.3.2. Chromogenic Assays

Compound #1 (10 mg) is dissolved in 50 ml of corn oil and the resultinggreen oil is maintained at room temperature under aerobic conditions forfour to seven months. During this time, the color of the solutionchanges from green to yellow to red, reflecting the progressive,increasing rancidity of the oil. It is noted that Compound #1, whenadded to continuously aerated chlorobenzene (a solvent that contains noreadily oxidizable groups, unlike vegetable oil), remains unchanged overtime.

6.4. Diagnostic, Prophylactic, or Therapeutic Applications

6.4.1. Prolongation of the Life Span of the Senescence Accelerated Mouse(SAM-P8)

SAM-P8 mice are available from Prof. Toshio Takeda (Kyoto University,Japan). The mice are housed under standard conditions at 25° C. with a12-hour light/dark cycle and allowed free access to water and a standarddiet. At 3 months of age, they were divided into four groups: two groups(12 male and 12 female mice) are used as control, and two groups aredesignated the experimental groups (13 male and 12 female mice). Theexperimental groups are given an azulenyl nitrone (either Compound 4 or26, 30 mg/kg, i.p.) daily and their body weight is measured. The controlgroups are sham injected with saline.

At the end of the study, it is observed that the azulenyl nitrones ofthe present invention prolong the life span of SAM-P8 mice significantly(i.e., by about 20-30%).

6.4.2. Delay of Senescence in Human Diploid Fibroblast Cells

IM-90 cells are obtained from the Coriell Institute for Medical Researchat population doubling level (PDL) 10.85. The population doublings (PDs)are calculated as log₂ (D/D₀), where D is the density of cells whenharvesting and D₀ is the density of cells when seeding. The stockcultures are split weekly and grown in 100-mm Corning tissue culturedishes containing 10 ml of Delbecco's modified Eagle's medium (DMEM)supplemented with 10% (vol/vol) dialyzed fetal bovine serum (Sigma).

To test the effect of ambient oxygen on the life span, cells arecultured in 25-cm² Corning flasks with 5 ml of medium. Early-passagecells are seeded at 0.1-0.3×10⁶ cells per flask and late-passage cellsare seeded at 0.5×10⁶ cells per flask. The flasks are gassed with amixture of 3% O₂ /5% CO₂ /92% N₂ or with a mixture of 20% O₂ /5% CO₂/75% N₂ for 30 sec, then plug-sealed, and incubated at 37° C. Thecultures are split after the cells reached confluence. Early-passagecells usually reach confluence in 5 or 6 days, and late-passage cells,even with increased seeding density, reach saturation density in 10-14days. At senescence, cells have not doubled for at least 21 days.

To determine the effect of azulenyl nitrone on the replicative life spanof cells, Compound 26 (stock - 50 mM in phosphate-buffered saline) isadded to culture medium at a final concentration of 200-1200 μM aftereach splitting. If cells are not split on day 7, the cells are fed withfresh medium containing Compound 26.

It is found that Compound 26 not only delays the onset of senescence,relative to untreated cells, but the azulenyl nitrone also rejuvenatesnear senescent cells in a dose-dependent manner.

6.4.3. Improvement of Cognitive Performance of Sprague-Dawley Rats

Forty male Sprague-Dawley Rats (aged 24-month-old) are divided into fivegroups of 8 rats each. Three groups are treated over a period of 3-5months with daily intraperitoneal (ip) injections of azulenyl nitrone 4at different dosages (5 mg/kg, 15 mg/kg and 30 mg/kg) in saline. Afourth group is treated with 15 mg/kg of the azulenyl nitrone for aperiod of 20 days only. The fifth group is treated with saline only andserves as a control.

One month after the stated treatment period, each group is tested in aMorris water maze. The rats are scored for their rates of acquisition(i.e., learning), memory retention, passive avoidance behavior, motoractivity, motor skill and any differences in their basal levels of brainlipid peroxidation. The latter may be gauged by TBAR formation. It isfound that, compared to the control group and the group treated for 20days only, the rats in the groups receiving 5, 15 and 30 mg/kg ofazulenyl nitrone 4 for at least three months exhibit better cognitiveperformance in terms of acquisition and memory retention, and arefurther found after 5 months of chronic treatment to have reduced levelsof brain lipid peroxidation.

This result demonstrates the effectiveness of an azulenyl nitrone of thepresent invention to inhibit the negative effects of free radical-basedaging on brain function and physiology.

6.4.4. Reduction in Multiple Organ Dysfunction and Cytokine Secretion

A saline solution of lipo-polysaccharide is administered to 16 maleSprague-Dawley Rats to induce organ dysfunction and the secretion of avariety of cytokines, including tumor necrosis factor-alpha (TNF-alpha),interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta).Thirty to forty-five minutes prior to LPS administration, half of therats are treated with intraperitoneal injections of azulenyl nitrone 1at varying dosages (5-100 mg/kg). Several markers are monitored,including serum levels of aspartate aminotransferase (AST) and alanineaminotransferase (ALT). The levels of AST and ALT are taken asindicators of LPs-induced liver damage. Also monitored are serum levelsof urea and creatinine, which indicate kidney damage induced by LPS.

It is found that a dose-dependent reduction in these liver and kidneydamage markers can be correlated to azulenyl nitrone administration. Itis also discovered that azulenyl nitrone 1 is able to prevent (i.e.,serve as a prophylatic against) LPS-induced pulmonary edema. Thisnitrone also exhibits some inhibition of both thrombocytopenia andleukopenia. Moreover, marked decreases in serum levels of LPS-stimulatedTNF-alpha, IL-1 alpha and IL-1 beta are observed.

6.4.5. Inhibition of Oxidative Modification of Cholesterol andTriglycerides of LDL

Albumin-free LDL is dialyzed against a buffer containing 50 mM borate atpH 9.0. Alternatively, a buffer containing 0.15M NaCl/5 mM Tris/1 mMCaCl₂ /0.1 mM EDTA at pH 7.4 can also be used. Dialysis is carried outunder an atmosphere of nitrogen at 4 degrees C. over a 48 h period, withup to six changes of buffer. Dialyzed LDL (0.5 mg protein/ml) isincubated with bee venom phospholipase A₂ (PLA₂, 3.3 units/ml) at 37degrees C. After 2 h, 26,000 units/ml of Soybean LO (SLO, type V, Sigma,St. Louis, Mo.) is added. Incubation is then continued with gentleshaking at 37 degrees C. under an ambient atmosphere.

To determine baseline levels of oxidative modification of LDL in theabsence of azulenyl nitrone, aliquots are taken at predetermined timeintervals over a 24 h period. Protein can be determined by the method ofLowry, H. O. et al., in J. Biol. Chem. (1951) 193:265 and the neutrallipid profile can be obtained by the method of Kuksis, A. et al., in J.Chromatogr. Sci. (1975)13:423.

The effect of azulenyl nitrone is examined by the addition of azulenylnitrone to the incubation mixture comprising LDL/PLA₂ at time 0 h atvarying final concentrations (0, 0.5, 1.0, 2.0, 5.0 and 10.0 mM).Incubation is then continued at 37 degrees C. for 2 h, followed by theaddition of SLO. After 2 h, the reaction is quenched by cooling theincubation tubes in ice water and the addition of 3 mM EDTA/0.05% (w/v)reduced glutathione under an argon atmosphere.

The results indicate that azulenyl compounds of the present inventioninhibit the oxidation of cholesteryl esters and triglycerides of LDL ina concentration-dependent, though not necessarily linear, manner.

The foregoing examples of preferred embodiments are provided simply toillustrate the present invention. Other embodiments of the presentinvention are apparent to one of ordinary skill in the art and areconsidered to fall within the scope and spirit of the present invention.Hence, the examples are not to be construed to limit the invention inany way, which invention is limited solely by the claims that follow.

6.4.6. In Vivo Diagnostic Applications

The ability of azulenyl nitrones to cross the blood brain barrier isdetermined by administering to a rodent the nitrone in a lipid (e.g.,lecithin, liposome, lipofectin, or lipofectamine) mixture and analyzingthe blood and brains for nitrone and/or aldehyde. As an example, miceare dosed at about 15 mg/kg iv with azulenyl nitrone 1 in aliposome-based mixture at a concentration of 1.5 mg/ml. Blood is sampledand the brains are perfused with saline. Afterward, the brains areremoved. The azulenyl nitrone 1 is extracted from the blood plasma andbrain tissue and measured. It is demonstrated that a higherconcentration of azulenyl nitrone is found in the brains of the micethan in their plasma and further demonstrates that 1 is able to crossthe blood brain barrier and is absorbed by brain tissue.

Furthermore, it is shown that when an ischemic event is induced inrodents a higher concentration of aldehyde by-product is detectable inthe brains of the rodents. As an example, several test and sham gerbilsare intraperitoneally (ip) injected with azulenyl nitrone 1 at 100 mg/kgin a liposome-based solution. Thirty minutes after injection, abilateral carotid occlusion (BCO) is induced on the test gerbils withsubsequent reperfusion. Measurements of the ratio of aldehyde to nitronein the hippocampus of the gerbils show a higher ratio for the BCOtreated test-gerbils. It is expected that the BCO treated gerbils wouldgenerated free radical species in their hippocampus due to the ischemicevent. A higher concentration of azulenyl aldehyde product is consistentwith the mechanism of azulenyl nitrone free radical trapping anddemonstrates the utility of these nitrones. Furthermore, it is shownthat the aldehyde product thus generated is predominately isolated tothe region of free radical formation, namely the brain tissue. Nosubstantial amount of azalenyl aldehyde product is observed in theblood.

6.4.7. In Vivo Neuroprotection

In this model the carotid arteries of gerbils are constricted surgicallyand after a predetermined period, the constriction is removed causingreperfusion and consequently the formation of oxygen free radicals. Thetest gerbils are dosed intraperitoneally at 100 mg/kg with azulenylnitrone 1 some time prior to the constriction and 100 mg/kgintraperitoneally some time after the constriction. After several daysof reperfusion, the hippocampal cells of the test and sham gerbils arecounted. As is expected, an 80% loss of cell viability is observed forthe sham gerbils, however, about twice as many viable cells are foundfor the azulenyl nitrone administered gerbils. Thus, the inventionserves as a neuroprotectant and reduces the infarct volume resultingfrom the ischemia and/or reperfusion.

6.4.8. Anti-Inflammation Topical Treatment

These compositions are in the form of a solution, a cream, a powder,gel, ointrnent, or lotion. They also constitute makeup or makeoverproducts or dermatological cakes containing the ingredients standard tothese types of compositions.

    ______________________________________                                        A cream is prepared as follows:                                               ______________________________________                                        azulenyl nitrone 34   1 to 0.25 g.                                            Titanium oxide        10 g.                                                   Red iron oxide        0.3 g.                                                  Yellow iron oxide     0.2 g.                                                  Brown iron oxide      0.4 g.                                                  Chestnut iron oxide   0.2 g.                                                  ______________________________________                                    

Several stearyl alcohols oxyethylenated with 33 mols. of:

    ______________________________________                                        Ethylene oxide        7 g.                                                    Propyl parahydroxybenzoate                                                                          0.2 g.                                                  Polyglycol stearate   6 g.                                                    Water, Q.S.P.         100 g.                                                  ______________________________________                                    

Other creams identical to that described immediately above are preparedby replacing azulenyl nitrone 34 with any of the previously mentionednitrone compounds.

A dermatological cleansing cake is prepared by mixing together thefollowing components:

    ______________________________________                                        Esters of sodium isothionate and                                                                    75 g.                                                   coprafatty acids (sold under the                                              tradename "IGEPON A" having the                                               formula R--COO--CH2--CH2--SO3--Na,                                            wherein R equals fatty acid deri-                                             vatives having 12-15 carbon atoms)                                            Lanolin derivatives   22.75 g.                                                azulenyl nitrone 4(1, acid Na salt)                                                                 0.75 g.                                                 ______________________________________                                    

Other dermatological cleansing cakes, identical to the above, areprepared by replacing azulenylnitrone 4 (1, acid Na salt) with any oneof the aforementioned active compounds.

A powder comprising the following mixture:

    ______________________________________                                        Talc                  99.6 g.                                                 Glycerine oleate      3.0 g.                                                  Isopropyl myristate   7.0 g.                                                  azulenyl nitrone 1    0.5 g.                                                  Perfume               2 cc.                                                   ______________________________________                                    

Other equally effective powder compositions identical to the above areprepared except that the active ingredient azulenyl nitrone 1 isreplaced by any of the other aforementioned active compounds.

A cream is made by dispersing 0.5 g of azulenyl nitrone 1 or 0.2 g of 34in 30.0 g of propylene glycol. The mixture is then homogenized into 97.4grams of finished cream, ointment or lotion following a modification ofany one of the procedures described in F. W. Martin et al, "Remington'sPharmaceutical Sciences", 14th Ed, Mack Publishing Co., Easton, Pa.1965.

6.5. Other Indications

The azulenyl nitrones of the present invention find htrther use in thetreatment of a variety of other ailments and conditions that aremediated by the inappropriate action of free radicals, including but notlimited to oxidative tissue damage, CNS spinal column damage andophthalmic disorders, progressive neuronal disorders, acute CNSoxidation in stroke, gradual CNS oxidation, migraines, gastriculceration, ulcers, certain aspects of diarrhea, gastritis, esophagitis,ileitis, ATP depletion in tissue, peripheral organ disease (such asatherosclerosis, bedsores, wounds and muscle overextension), shock,memory disorders, including short term memory loss. The compounds of theinvention can also be. useful as analgesics, in particular, as anon-steroidal anti-inflammatory drug (or NSAID). For further informationon the indications listed above, the interested reader is referred toU.S. Pat. Nos. Re 35112, 5,025,032, 5,508,305, 5,488,148, 5,036,097,5,475,032, 5,292,746 and 5,405,874, the disclosures of each of which areincorporated by reference herein.

What is claimed is:
 1. A compound of the formula: ##STR12## in which R₁may be a hydrogen, a linear or branched alkyl group comprising 1-6carbon atoms, or an aryl group comprising 6-10 carbon atoms;R₂ may be alinear or branched alkyl group comprising 1-6 carbon atoms, or an arylgroup comprising 6-10 carbon atoms; R₃ may be a hydrogen, or a linear orbranched alkyl group comprising 1-6 carbon atoms; R₄ may be a hydrogen,or a linear or branched alkyl group comprising 1-6 carbon atoms; R' maybe a linear or branched alkyl group comprising 1-6 carbon atoms; W maybe a linear or branched alkyl group comprising 1-6 carbon atoms, an arylgroup comprising 6-10 carbon atoms, or an electron-withdrawing group; nmay be 0, 1, or 2 (if n is 2, each W may be the same as or differentfrom one another); m may be 0, 1, 2, or 3 (if m is 2 or 3, each R' maybe the same as or different from one another); o may be 1 or 2 (if o is2, each R₁ and R₂ may be the same as or different from one another); pmay be 0, 1, or 2 (if p is 2, each R₃ and R₄ may be the same as ordifferent from one another) or a salt thereof.
 2. The compound of claim1 in which p is
 0. 3. The compound of claim 1 in which n is
 1. 4. Thecompound of claim 1 in which m is 1 or
 2. 5. The compound of claim 1 inwhich the groups R₁, R₃, and R₄ are all hydrogen.
 6. The compound ofclaim 4 in which at least one R' is a methyl group.
 7. The compound ofclaim 4 in which at least one R' is an ethyl or isopropyl group.
 8. Thecompound of claim 1 in which the group R₂ is a tert-butyl group.
 9. Thecompound of claim 1 in which W is an electron-withdrawing group.
 10. Thecompound of claim 1 in which the group (CHR₃ ═CR₄)_(p) C(R₁)═N⁺ (R₂)O⁻is at the 1-position when the group W is at the 3-position.
 11. Thecompound of claim 10 in which m is 2 and the groups R' are at the 4- and7-positions.
 12. The compound of claim 9 in which the group W may be acarboxylic acid, carboxylic acid ester, sulfonic acid, sulfonic acidester, ketone, halogen, cyano, nitro, nitroso, aldehyde, phosphoricacid, phosphoric acid ester, sulfoxide, sulfone, or a salt thereof. 13.The compound of claim 12 in which the group W is a trifluoroacetylgroup.
 14. The compound of claim 1 in which o is
 1. 15. The compound ofclaim 1 which is 2-methyl[1-(3-carboxylicacid-7-isopropyl-4-methyl)azulenylmethylene]-2-propanamine N-oxide, itsester, amide, or salt.
 16. The compound of claim 1 which is2-methyl[1-(3-carboethoxy-7-isopropyl-4-methyl)azulenylmethylene]-2-propanamineN-oxide.
 17. The compound of claim 1 which is 2-methyl[1-(3-sulfonicacid-7-isopropyl-4-methyl)azulenylmethylene]-2-propanamine N-oxide, itsester, amide, or salt.
 18. The compound of claim 1 which is2-methyl[1-(3-methylsulfonyl-7-isopropyl-4-methyl)azulenylmethylene]-2-propanamineN-oxide.
 19. A compound of the formula: ##STR13## in which R₁ may be ahydrogen, a linear or branched alkyl group comprising 1-6 carbon atoms,or an aryl group comprising 6-10 carbon atoms;R₂ may be a linear orbranched alkyl group comprising 1-6 carbon atoms, or an aryl groupcomprising 6-10 carbon atoms; R' may be a linear or branched alkyl groupcomprising 1-6 carbon atoms; W may be a linear or branched alkyl groupcomprising 1-6 carbon atoms, an aryl group comprising 6-10 carbon atoms,or an electron-withdrawing group; m may be 0, 1, 2, or 3 (if m is 2 or3, each R' may be the same as or different from one another) or a saltthereof.
 20. The compound of claim 19 in the form of its metal salt. 21.The compound of claim 20 in which said metal salt is an alkali oralkaline-earth metal salt.
 22. The compound of claim 19 in the form ofits ammonium or tetraalkylammonium salt.
 23. The compound of claim 19which appears green to the naked eye.
 24. The compound of claim 1 whichis 1,3-bis(2-methyl-2-propanamine N-oxide)azulenyldimethylene.
 25. Thecompound of claim 1 which is 1,3-bis(2-methyl-2-propanamineN-oxide)-7-isopropyl-4-methylazulen-yldimethylene.
 26. A pharmaceuticalcomposition for alleviating the ill effects of a pathologic conditionmediated or initiated by a reactive free radical, said compositioncomprising an effective amount of the compound of claim 19 and apharmaceutically acceptable carrier.
 27. A composition comprising acompound of the formula: ##STR14## in which R₁ may be a hydrogen, alinear or branched alkyl group comprising 1-6 carbon atoms, or an arylgroup comprising 6-10 carbon atoms;R₂ may be a linear or branched alkylgroup comprising 1-6 carbon atoms, or an aryl group comprising 6-10carbon atoms; R₃ may be a hydrogen, or a linear or branched alkyl groupcomprising 1-6 carbon atoms; R₄ may be a hydrogen, or a linear orbranched alkyl group comprising 1-6 carbon atoms; R' may be a linear orbranched alkyl group comprising 1-6 carbon atoms; W may be a linear orbranched alkyl group comprising 1-6 carbon atoms, an aryl groupcomprising 6-10 carbon atoms, or an electron-withdrawing group; n may be0, 1, or 2 (if n is 2, each W may be the same as or different from oneanother); m may be 0, 1, 2, or 3 (if m is 2 or 3, each R' may be thesame as or different from one another); o may be 1 or 2 (if o is 2, eachR₁ and R₂ may be the same as or different from one another); p may be 0,1, or 2 (if p is 2, each R₃ and R₄ may be the same as or different fromone another) or a salt thereof; and a carrier.
 28. A process for makingan azulenyl nitrone comprising:(a) providing an azulene; (b) introducinga an acyl group to said azulene at a position that is to bear a nitronegroup; (c) converting said acyl group to a nitrone group to provide anazulenyl nitrone.
 29. A compound of formula ##STR15## in which R₁ may bea hydrogen, a linear or branched alkyl group comprising 1-6 carbonatoms, or an aryl group comprising 6-10 carbon atoms;R₂ may be a linearor branched alkyl group comprising 1-6 carbon atoms, or an aryl groupcomprising 6-10 carbon atoms; R₃ may be a hydrogen, or a linear orbranched alkyl group comprising 1-6 carbon atoms; R₄ may be a hydrogen,or a linear or branched alkyl group comprising 1-6 carbon atoms; R₅ maybe a hydrogen, or a linear or branched alkyl group comprising 1-6 carbonatoms; R₆ may be a hydrogen, or a linear or branched alkyl groupcomprising 1-6 carbon atoms; R' may be a linear or branched alkyl groupcomprising 1-6 carbon atoms; W may be a linear or branched alkyl groupcomprising 1-6 carbon atoms, an aryl group comprising 6-10 carbon atoms,or an electron-withdrawing group; n may be 0, 1, or 2 (if n is 2, each Wmay be the same as or different from one another); m may be 0, 1, 2, or3 (if m is 2 or 3, each R' may be the same as or different from oneanother); o may be 1 or 2 (if o is 2, each R₁ and R₂ may be the same asor different from one another); p may be 0, 1, or 2 (if p is 2, each R₃and R₄ may be the same as or different from one another); q may be 0, 1,2, 3 or 4 (if q is greater than 1, R₅ and R₆ may be the same as ordifferent from one another) or a salt thereof.
 30. A method ofalleviating the ill effects of ischemia or reperfusion injury in asubject comprising administering to said subject an effective amount ofa compound of claim 1 or
 19. 31. A method of alleviating the ill efrectsof Acute Respiratory Distress Syndrome (ARDS) in a subject comprisingadministering to said subject an effective amount of the compound ofclaim 1 or
 19. 32. A method of alleviating the ill effects of aging orsenescence in a subject comprising administering to said subject aneffective amount of the compound of claim 1 or claim 19.